Enhanced homologous recombination mediated by lambda recombination proteins

ABSTRACT

Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.

PRIORITY CLAIM

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/366,044, filed Feb. 12, 2003, which claims priority toInternational Application No. PCT/US01/25507, filed Aug. 14, 2001, whichclaims the benefit of U.S. Provisional Application No. 60/225,164, filedAug. 14, 2000, and claims the benefit of U.S. Provisional ApplicationNo. 60/271,632, filed Feb. 26, 2001. All of the prior applications areincorporated by reference herein in their entirety.

FIELD

[0002] The present disclosure relates to methods to enhance homologousrecombination in bacteria and eukaryotic cells using recombinationproteins, such as those derived from bacteriophage lambda. It alsorelates to methods for modifying genomic DNA in bacterial artificialchromosomes (BACs) and to subcloning of genomic DNA from BACs intomulticopy plasmids.

BACKGROUND OF THE INVENTION

[0003] Concerted use of restriction endonucleases and DNA ligases allowsin vitro recombination of DNA sequences. The recombinant DNA generatedby restriction and ligation may be amplified in an appropriatemicroorganism such as E. coli, and used for diverse purposes includinggene therapy. However, the restriction-ligation approach has twopractical limitations: first, DNA molecules can be precisely combinedonly if convenient restriction sites are available; second, becauseuseful restriction sites often repeat in a long stretch of DNA, the sizeof DNA fragments that can be manipulated are limited, usually to lessthan about 25 kilobases.

[0004] Homologous recombination, generally defined as an exchangebetween homologous segments anywhere along a length of two DNAmolecules, provides an alternative method for engineering DNA. Ingenerating recombinant DNA with homologous recombination, amicroorganism such as E. coli, or a eukaryotic cell such as a yeast orvertebrate cell, is transformed with exogenous DNA. The center of theexogenous DNA contains the desired transgene, whereas each flankcontains a segment of homology with the cell's DNA. The exogenous DNA isintroduced into the cell with standard techniques such aselectroporation or calcium phosphate-mediated transfection, andrecombines into the cell's DNA, for example with the assistance ofrecombination-promoting proteins in the cell.

[0005] In generating recombinant DNA by homologous recombination, it isoften advantageous to work with short linear segments of DNA. Forexample, a mutation may be introduced into a linear segment of DNA usingpolymerase chain reaction (PCR) techniques. Under proper circumstances,the mutation may then be introduced into cellular DNA by homologousrecombination. Such short linear DNA segments can transform yeast, butsubsequent manipulation of recombinant DNA in yeast is laborious. It isgenerally easier to work in bacteria, but linear DNA fragments do notreadily transform bacteria (due in part to degradation by bacterialexonucleases). Accordingly, recombinants are rare, require specialpoorly-growing strains (such as RecBCD- mutant strains) and generallyrequire thousands of base pairs of homology. Thus, improved methods ofpromoting homologous recombination in bacteria are needed.

[0006] In eukaryotic cells, targeted homologous recombination provides abasis for targeting and altering essentially any desired sequence in aduplex DNA molecule, such as targeting a DNA sequence in a chromosomefor replacement by another sequence. The approach may be useful fortreating human genetic diseases.

[0007] Homologous recombination has been used to create knock-outmutants and transgenic animals, and thereby has played a criticallyimportant role in understanding gene function. Transgenic animals areorganisms that contain stably integrated copies of genes or geneconstructs derived from another species in the chromosome of thetransgenic animal. These animals can be generated by introducing clonedDNA constructs of the foreign genes into totipotent cells by a varietyof methods, including homologous recombination.

[0008] Currently, methods for producing transgenics have been performedon totipotent embryonic stem cells (ES) and with fertilized zygotes. EScells have an advantage in that large numbers of cells can bemanipulated in vitro before they are used to generate transgenics.Alternatively, DNA can also be introduced into fertilized oocytes bymicro-injection into pronuclei, or injection into the germiline oforganisms including C. elegans or Drosophila species.

[0009] Several methods have been developed to detect and/or select fortargeted site-specific recombinants between vector DNA and the targethomologous chromosomal sequence (Capecchi, Science 244:1288, 1989).Cells that exhibit a specific phenotype after recombination, such asoccurs with alteration of the hypoxanthine phosphoribosyl transferase(hprt) gene, can be obtained by direct selection on the appropriategrowth medium. Alternatively, a selectable marker such as neomycinresistance can be incorporated into a vector under promoter control, andsuccessful transfection can be scored by selecting G418-resistant cells(Joyner et al., Nature 338:153, 1989). Numerous other selectionprocedures have been described (Jasin and Berg, Genes and Development2:1353, 1988; Doetschman et al., Proc. Natl. Acad. Sci. U.S.A. 85:8583,1988; Dorini et al., Science 243:1357, 1989; Itzhaki and Porter, Nucl.Acids Res. 19:3835, 1991). Unfortunately, exogenous sequencestransferred into eukaryotic cells undergo homologous recombination onlyat very low frequencies, even when very long homology regions arepresent (Koller et al., Proc. Natl. Acad. Sci. U.S.A., 88:10730, 1991,and Snouwaert et al., Science 257:1083, 1992). Thus, large numbers ofcells must be transfected, selected, and screened in order to generate acorrectly targeted homologous recombinant.

SUMMARY OF THE DISCLOSURE

[0010] The present disclosure provides methods for cloning DNA moleculesin cells having DNA encoding lambda recombinases operably linked to ade-repressible promoter, for example the lambda pL promoter. The pLpromoter is activated, for example by temperature shift, thereby leadingto expression of lambda recombinases. The lambda recombinases promotehomologous recombination between nucleic acids in the cell. The nucleicacids undergoing recombination may be intrachromosomal, or may beextrachromosomal, for example in a bacterial artificial chromosome.

[0011] The present disclosure also provides methods for inducinghomologous recombination using single-stranded DNA molecules, byintroducing into the cell DNA capable of undergoing homologousrecombination, and a single-stranded DNA binding polypeptide capable ofpromoting homologous recombination. Such single-stranded DNA bindingpolypeptides include lambda Beta, RecT, P22 Erf, and Rad52, as well asfunctional fragments and variants of single-stranded DNA bindingpolypeptides.

[0012] The present disclosure also provides bacterial cells that promoteefficient homologous recombination. These bacterial cells contain one ormore genes or promoters from a defective lambda prophage within thebacterial chromosome.

[0013] The disclosure also provides methods for altering eukaryoticgenes by expressing recombinases operably linked to a de-repressiblepromoter in bacterial cells. Eukaryotic genes thus modified can be usedto modify eukaryotic cells, for example to generate transgenic orknockout animals.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 is a linear depiction of the defective lambda prophage asit is integrated on the E. coli chromosome. Prophage genes are indicatedby the solid line, and E. coli genes by broken lines. The coding regionsfor the lambda recombination genes exo, bet and gam are approximately inthe center of the defective prophage. Lambda genes cro through attR,deleted from the defective prophage, are enclosed within parenthesestogether with the E. coli bioA gene to indicate their deletion.

[0015]FIG. 2A is a schematic diagram showing classical recombinanttechnology, and FIG. 2B is a schematic diagram showing “recombineering”using homologous recombination, as disclosed herein.

[0016]FIG. 3 is a graph that shows the effect of induction time of thelambda proteins Beta, Exo, and Gam on recombination efficiency. A linearchloramphenicol resistance cassette was used to target prophage genes,and was electroporated into cells after the cells had been heated to 42°C. for the time indicated. Time of induction (temperature shift to 42°C.) is plotted on the x-axis, against number ofchloramphenicol-resistant recombinants obtained on the y-axis.

[0017]FIG. 4 is a graph that shows the effect of linear DNA amount onrecombination. The E. Coli strain DY330 was induced at 42° C. for 15minutes, and DNA encoding a linear chloramphenicol resistance cassette(<cat>) at the indicated concentration was electroporated into cells.DNA concentration encoding <cat> is plotted on the x-axis, againstnumber of chloramphenicol-resistant recombinants obtained on the y-axis.

[0018]FIG. 5 is a graph which shows the effect of homologous arm lengthon recombination in E. coli cells induced for expression of lambdarecombination proteins Beta, Exo, and Gam. A chloramphenicol resistancecassette was synthesized with homologous arms of the indicated length atits 5′ and 3′ end. Homologous arm length was varied from 0 to 1,000 basepairs. Length of homologous arms is plotted on the x-axis, againstnumber of chloramphenicol-resistant recombinants (log scale) on they-axis.

[0019]FIG. 6 is a linear depiction of the modified defective lambdaprophage as integrated on the E. coli chromosome of DY380, EL250, andEL350 cells. This figure illustrates that the defective prophages usedfor BAC engineering contain the λ genes from cI857 to int. P_(L) (or pL)and P_(R) denote the lambda left and right promoters, respectively. Thegam and red genes, exo and bet are under the control of P_(L), which isrepressed by the temperature-sensitive repressor, cI857 at 32° C. andderepressed at 42° C. tet replaces the segment from cro-bioA in DY380cells. The araC-P_(BAD)flpe cassette or the araC-P_(BAD)cre cassettereplaces the segment from cro-bioA in EL250 or EL350 cells,respectively. The promoter of the araBAD operon (P_(BAD)), which can beinduced by L-arabinose, controls the expression of the flpe or Cregenes. Thick black lines designate the prophage while thin linesrepresent E. coli sequence. < > defines the ends of the cro-bioA regionthat was replaced with tet, araC-P_(BAD) flpe, or araC-P_(BAD) cre.

[0020]FIG. 7 illustrates a strategy for BAC engineering. This figureillustrates the relative position of the Eno2 gene in the fullysequenced 250-kbp BAC, 284H12 and the different steps used to introduceCre into the last exon of Eno2. In the targeting cassette, frt sites aredenoted by ellipses, the kan gene by a red rectangle and the GFPcrefusion gene by a blue rectangle. The green boxes represent Eno2 exons.

[0021]FIG. 8 is a schematic diagram illustrating the use of the gaprepair to subclone fragments as large as 80 kbp from BACs. FIG. 8A showsas short thick black arrows the location of the 5′ homologies on theamplification primers used to amplify pBR322 for subcloning by gaprepair. Each primer also contains 20 nt segments at its 3′ end to primepBR322. NotI and SalI cleavage sites were included in these primers tofacilitate release of the subcloned fragments from the plasmid backbone.The location of SpeI restriction sites near Eno2 is also shown (“S”).SpeI restriction sites are not present on the linear amplified pBR322vector. FIG. 8B shows an intermediate step in gap repair, pairingbetween a typical amplified pBR322 targeting cassette and the modifiedEno2 BAC. Ap, amp resistance gene; ORI, origin of replication.

[0022]FIG. 9 is a schematic diagram of a defective λ prophage. Thedefective prophage DY380 expressing the λ Exo, Beta, and Gam functionsis shown with the genes under P_(L) promoter control and the temperaturesensitive repressor, CI857. Advantages and disadvantages of the systemsare described. The genes encoding Cre and Flpe are present on otherderivatives of DY380 (EL250 and EL350) and replace the tet gene asshown.

[0023]FIG. 10 is a schematic diagram of in vivo cloning by recombinationusing gap repair of a linear vector plasmid. The method of in vivocloning uses two linear DNAs, a vector and a target DNA, that havehomologies to each other at their ends. Both are electroporated intocompetent cells to allow recombination and gap repair of the plasmid.The linear vector is made in a similar way to that shown in FIG. 8.

[0024]FIG. 11 is a schematic diagram of in vivo retrieving of DNA fromBAC clones. Retrieving of segments up to 80 kbp from BACs intoPCR-amplified vectors has been possible using recombineering techniquesdisclosed herein. Here only the plasmid is linearized and transformedinto a recombination competent cell containing the BAC. Recombinationoccurs between homologies on the end of the linear vector and the BAC.This method eliminates standard cloning technology from the BAC, andimportantly, the cloned segment is never replicated in vitro, therebyreducing the chance of extraneous changes in the sequence.

[0025]FIG. 12 is a schematic diagram of a mini-lambda DNA circle. Thislambda DNA element is not a plasmid and lacks any replication originactivity. It does contain the lambda cI85 7 repressor and the pL operonthat the repressor controls. It also contains a cassette encoding a drugmarker, in this case the tet genes. This DNA when transformed into moststrains including the BAC strains makes Int protein allowing integrationof the circular DNA at the λ attachment site on the bacterialchromosome, and cI857 repressor to allow repression of pL. Theintegrated mini-lambda is stable but able to be induced at 42° C. toactivate Gam, Beta, and Exo expression to make the cell recombinationcompetent.

[0026]FIG. 13 is a schematic diagram of recombination of ssDNA into thegenome. When ssDNA is electroporated into cells, it is bound by Betaprotein and recombined into the genome or into a BAC plasmid byhomology. Evidence suggests that Beta-bound ssDNA anneals to its ssDNAcomplement at the replication fork. The strand of DNA corresponding tothat made by lagging strand synthesis is most recombinogenic suggestingthat Beta simply anneals the ssDNA to a gap caused by DNA replication.

[0027]FIG. 14 is a schematic diagram of subcloning a DNA fragment from aBAC into pBluescript (pSK⁺) by gap repair with short homology arms viarecombineering. Primers that have 20 bp of homology (arrows) topBluescript (circle) at their 3′ end, and 50 bp (dark area) of homologyat their 5′ ends to one of two ends of the BAC DNA to be subcloned(thinner areas, exon 4 in center), are used to amplify pBluescript. ThePCR-amplified, linearized, pBluescript containing the two homology armsis then transformed into recombination-competent cells that carry theBAC. Gap-repaired plasmids are selected by their ampicillin resistance.The black bar denotes the location of Evi9 exon 4.

[0028]FIG. 15 is a schematic diagram of an improved procedure forsubcloning DNA from BACs and for constructing cko-targeting vectors. Thehomology arms used for gap repair (subcloning) and for targeting, arePCR-amplified from BAC DNA. The two-homology arms (arrow, segmentsending with AB or YZ, homologies indicated by light lines to plasmid),amplified using primers A and B or primers Y and Z, were cloned into aMC1TK-containing plasmid, to generate the gap repair (retrieval) plasmidfor subcloning. The gap repair plasmid was linearized with HindIII tocreate a DNA double strand break for gap repair. A mini-targeting vectorwas constructed by ligating together the two PCR products generated byamplification of BAC DNA with primers C and D (segment indicated asending with CD) or primers E and F (segments indicated as ending withEF), a floxed Neo selection cassette (black arrow: LoxP site), andpBluescript. A Bg/II restriction site was included in the mini-targetingvector for diagnosing gene targeting in ES cells. The black arrowsdenote LoxP sites. The targeting cassette was excised by NotI and Salldigestion, or by PCR amplification, using primers C and F. Thegap-repaired plasmid, and the excised targeting cassette, wereco-transformed into recombination-competent DY380 or EL350 cells. Therecombinants had a floxed Neo cassette inserted between primers D and Eand can be selected on kanamycin plates. The Neo cassette was excisedwith Cre recombinase, leaving a single LoxP site at the targeted locus(see FIG. 16). Similarly, a Neo selection cassette can be insertedbetween primers H and I using homology arms amplified by primers G, H(segment indicated as ending with GH), and I, J (segments indicated asending with IJ).

[0029]FIGS. 16A and 16B are sets of schematic diagrams and a digitalimage of the construction of an Evi9 conditional knockout allele. FIG.16A is a set of schematic diagrams of the 11.0 kb genomic DNA fragmentcontaining Evi9 exon 4 was subcloned from BAC-A12 using gap repair.EcoRV digestion of the gap-repaired plasmid generates 7.6 kb and 8.8 kbfragments. The 7.6 kb fragment contains Evi9 exon 4 sequences, while the8.8 kb fragment, common to all lanes contains plasmid sequences and Evi9sequences located upstream of exon 4. The floxed Neo cassette of PL452was targeted upstream of Evi9 exon 4. In the targeted plasmid, the 7.6kb EcoRV fragment increases in size to 9.6 kb due to the addition of thefloxed Neo cassette. Excision of the floxed Neo cassette leaves behind asingle LoxP (black arrow) at the targeted locus, and the normal EcoRVdigestion pattern is restored. Next, the PL451 selection cassettecontaining the Neo gene flanked by frt sites (grey arrow) and adownstream LoxP, was targeted downstream of Evi9 exon 4. The PL451selection cassette contains an EcoRV site, which results in theproduction of 6.5 kb and 3.1 kb fragments following EcoRV digestion.This is the Evi9 cko-targeting vector. To test the functionality of thefrt sites in the cko-targeting vector, the PL451 selection cassette wasexcised from the cko-targeting vector by FLP recombinase followingelectroporation into EL250 cells. This reduces the size of the 6.5 kbEcoRV fragment to 4.5 kb. Finally, electroporation of the cko-targetingcassette into EL350 cells expressing Cre recombinase excises the entireDNA between the two-LoxP sites, creating a 4.6 kb EcoRV fragment. FIG.16B is a digital image of EcoRV-digestion patterns of the plasmids atevery stage of the targeting vector construction.

[0030]FIG. 17A and 17B are a schematic diagram and digital image showingthe identification of correctly targeted ES cell clones. FIG. 17A is aschematic diagram of homologous recombination between the Evi9cko-targeting vector and the Evi9 genomic locus. Correctly targeted EScells (cko allele) have a 5.5 kb Bg/II band, in addition to an 18.1 kbwild type band, following hybridization with the 5′ probe. These ckoclones also have a 6.3 kb EcoRV-targeted band, as well as a 7.3 kb wildtype band, following hybridization with the 3′ probe. FIG. 17B is adigital image of a Southern blot analysis of the ES cell clones. The 5′probe was used in the left panel and a 3′ probe was used in the rightpanel. wt: wild type ES clones, cko: conditional knockout ES clones.

[0031]FIG. 18 is a flow chart of making a conditional knockout vectorbased on recombineering.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0032] There exists a need in the art for methods of precisely andefficiently altering predetermined endogenous genetic sequences byhomologous recombination in vivo. There independently exists a need inthe art for high-efficiency gene targeting, so as to avoid complex invitro or in vivo selection protocols. Methods are disclosed herein forcloning DNA molecules in cells using homologous recombination mediatedby lambda recombinases and similar proteins.

[0033] One such method uses a cell having DNA encoding functional Betaand optionally Exo, and Gam, or functional fragments or variantsthereof, operably linked to the a de-repressible promoter (such as, butnot limited to, the pL promoter). De-repression of the de-repressiblepromoter (e.g. the induction of transcription from the pL promoter byinactivation of cI) induces expression of exo, bet and gam and in someembodiments may be selectively activated for this purpose. A nucleicacid (such as a polynucleotide which is homologous to a target DNAsequence) capable of undergoing homologous recombination is introducedinto the cell, and cells in which homologous recombination has occurredare either selected or found by direct screening of cells. In particularembodiments, the nucleic acid introduced into the cell may be doublestrand DNA, or DNA with 5′ overhangs.

[0034] In additional particular embodiments, at least 1 in 5000 cellscontain DNA in which homologous recombination has occurred. In furtherembodiments, at least 1 in 1,000 cells, or 1 in 500 cells, 1 in 100cells, or 1 in 20 cells contain DNA in which homologous recombinationhas occurred.

[0035] The cell may be a eukaryotic cell, or a prokaryotic cell, such asa bacterial cell, for example an E. coli strain. The DNA encoding thelambda recombination proteins and pL promoter may be intrachromosomal orextrachromosomal. Similarly, the target DNA sequence may beintrachromosomal or extrachromosomal; for example, the target DNAsequence may be found in a chromosome of the cell or a plasmid(including derivatives of co1E1, pSC101, p15A and shuttle vectors thatreplicate in both bacteria and eucaryotic cells), bacterial artificialchromosome, P1 artificial chromosome, yeast artificial chromosome,cosmid or the like.

[0036] In additional particular embodiments, the nucleic acid introducedinto the cell may be double-stranded DNA or DNA with a 5′ overhang, andmay include a positive or negative selectable marker. The introducednucleic acid may alter the function of a nucleic acid sequence such as agene in the cell, or add a gene to the DNA of the cell. The cell may betreated to enhance macromolecular uptake, for example usingelectroporation, calcium phosphate-DNA coprecipitation, liposomemediated transfer, or other suitable methods. In other particularembodiments, the method may produce homologous recombination that altersthe function of a gene in the cell, or adds a gene to the cell.

[0037] In further particular embodiments, the cell may be treated toenhance macromolecular uptake, for example with electroporation, calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, microinjection, liposome fusion,lipofection, protoplast fusion, inactivated adenovirus-mediatedtransfer, HVJ-liposome mediated transfer, and biolistics.

[0038] Another such method that meets one or more of the above-mentionedneeds includes methods that introduce into the cell DNA capable ofundergoing homologous recombination and a ssDNA binding polypeptidecapable of promoting homologous recombination. In particularembodiments, the DNA is ssDNA or DNA having 3′ overhangs.

[0039] The single stranded DNA (ssDNA) binding polypeptide is a type ofDNA binding polypeptide which mediates double strand break repairhomologous recombination by binding to ssDNA or a 3′ overhang in dsDNAand promoting recombination. It promotes recombination by annealing thebound ssDNA to its complement in the cell. Examples of such ssDNAbinding polypeptide include lambda Beta, E. coli RecT, Erf ofbacteriophage P22, and Rad52. The ssDNA binding polypeptide may beintroduced as a nucleic acid. For example, a nucleic acid that expressesthe ssDNA binding polypeptide is introduced into a cell, such as aeukaryotic cell. Expression of the ssDNA binding polypeptide from anucleic acid may be induced, for example, by activation of an induciblepromoter. In particular embodiments, the nucleic acid may furtherinclude lambda exo and gam, and the inducible promoter may be the lambdapL promoter. In other embodiments, the ssDNA binding polypeptide isintroduced into the cell as a polypeptide.

[0040] The cell used in methods disclosed herein may be a bacterial cellsuch as an E. coli strain, or a eukaryotic cell such as a mammaliancell, a stem cell, or virtually any other eukaryotic cell type. The DNAused in the method may be a single oligonucleotide sequence, or may betwo or more overlapping sequences, for example with more than 10, ormore than 20 base pairs of complementary overlap at either their 5′ or3′ termini (in specific examples of the 5′ case, the nucleic acidincludes exo and bet nucleic acid sequences). The DNA may comprise aselectable marker, and homologous recombination with the ssDNA mayconfer a selectable phenotype upon the cell. In particular embodiments,the cell may be treated to enhance macromolecular uptake, such as withelectroporation, calcium phosphate-DNA coprecipitation, liposomemediated transfer, or other suitable methods. The effect of homologousrecombination may be to alter the function of a gene in the cell, or adda gene to the cell.

[0041] In particular examples, the ssDNA is used in an amount of about0.01 μM to about 10 mM; or from about 0.1 μM to about 1 mM; or fromabout 1 μM to about 100 μM. In other examples, the ssDNA bindingpolypeptide is used in an amount of 0.001 μM to about 0.01 μM, or fromabout 0.01 μM to about 10 mM; or from about 0.1 μM to about 1 mM; orfrom about 1 μM to about 100 μM.

[0042] Also disclosed are bacterial cells that may be useful inpracticing the disclosed methods. These include bacterial cellsharboring a defective lambda prophage of genotype λcI857 Δ(cro-bioA). Inparticular examples, the bacterial cells may have a selectable marker,such as an antibiotic resistance marker, upstream of the cI857 gene. Insome particular examples, the disclosed bacterial cells include aninducible promoter upstream of the cI857 gene, which may be operablyconnected to a gene encoding a recombinase, such as flp, flpe, or Cre,or a gene encoding functional fragments or variants of theserecombinases. In other particular examples, the bacterial cells maycontain a bacterial artificial chromosome, which may have a selectablemarker, LoxP, and/or frt sites. In particular embodiments, theselectable marker on the bacterial artificial chromosome is excisable bya recombinase. In other particular examples, the bacterial artificialchromosome may have at least one exon or at least one intron of amammalian gene.

[0043] The disclosure also includes methods of altering eukaryotic genesby expressing in a bacterial cell an intrachromosomal gene encoding arecombinase operably linked to a pL promoter. The bacterial cell alsoincludes an extrachromosomal eukaryotic gene or gene fragment (having atleast one intron or at least one exon of a eukaryotic gene). A nucleicacid capable of undergoing homologous recombination with the eukaryoticgene is introduced into the bacterial cell, and the nucleic acidundergoes homologous recombination with the eukaryotic gene or genefragment. In a particular embodiment, the nucleic acid undergoeshomologous recombination with a targeting frequency of at least about 1in 1,000.

[0044] In one embodiment, the expressed recombinase is a double strandbreak repair recombinase, such as lambda Beta or other single-strandedDNA binding protein; lambda Exo, or lambda Gam. In another embodiment,the extrachromosomal eukaryotic gene or gene fragment may be located ona bacterial artificial chromosome, yeast artificial chromosome, P1artificial chromosome, plasmid or cosmid. In yet another embodiment, theeukaryotic gene or gene fragment is derived from a mammalian organism,such as a mouse or human.

[0045] In several additional embodiments, the nucleic acid undergoinghomologous recombination may encode a recombinase, functional fragmentsor variants of a recombinase, or an epitope tag.

[0046] Also disclosed are methods of altering intrachromosomal DNA of aeukaryotic cell. In these methods, an altered eukaryotic gene or genefragment is introduced into the eukaryotic cell. The introducedeukaryotic gene or gene fragment has been altered by homologousrecombination using the methods of this disclosure.

[0047] For example, extrachromosomal DNA including the eukaryotic. geneor gene fragment is introduced into a bacterial cell having anintrachromosomal gene encoding a recombinase operably linked to ade-repressible promoter. The bacterial cell is then induced to expressthe recombinase. A nucleic acid molecule capable of undergoinghomologous recombination with the eukaryotic gene or gene fragment isintroduced into the bacterial cell. The eukaryotic gene or gene fragmentundergoes homologous recombination with the nucleic acid, and alteredeukaryotic gene or gene fragment may then be isolated and introducedinto a eukaryotic cell.

[0048] In one embodiment, the eukaryotic gene or gene fragmentintroduced into the eukaryotic cell is located on a bacterial artificialchromosome. The eukaryotic gene or gene fragment is capable ofundergoing homologous recombination with a target gene in the cell,thereby altering the nucleic acid sequence of the eukaryotic cell'sintrachromosomal DNA. In specific, non-limiting examples, the eukaryoticcell is a mammalian cell, an embryonic stem cell, or a zygote.

[0049] Also disclosed are mutant mammals which have had one or more oftheir genes altered by homologous recombination with a bacterialartificial chromosome carrying a eukaryotic gene or gene fragment thathas been altered by the disclosed methods. The gene alteration canintroduce a recombinase into the mutant mammal, such as a site-specificrecombinase.

[0050] A mobilizable lambda DNA is also disclosed herein that isisolated as a mini-lambda prophage. The mobilizable lambda DNA can betransformed into any bacterial strain of interest. The lambda DNAintegrates into the bacterial chromosome to generate a defectiveprophage that expresses the recombinase.

[0051] The present disclosures provide methods of enhancing theefficiency of homologous recombination. The disclosures will be betterunderstood by reference to the following explanation of terms used anddetailed description of methods for carrying out the invention.

[0052] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

[0053] I. Terms

[0054] 3′ overhang: Two nucleic acid sequences which when annealed arepartially double-stranded and partially single-stranded. Thesingle-stranded end or ends extend away from the double-stranded segmentin a 5′ to 3′ direction.

[0055] 5′ overhang: Two nucleic acid sequences which when annealed arepartially double-stranded and partially single-stranded. Thesingle-stranded end or ends extend away from the double-stranded segmentin a 3′ to 5′ direction.

[0056] Antibiotic Resistance Cassette: A nucleic acid sequence encodinga selectable marker which confers resistance to that antibiotic in ahost cell in which the nucleic acid is translated. Examples ofantibiotic resistance cassettes include, but are not limited to:kanamycin, ampicillin, tetracycline, chloramphenicol, neomycin,hygromycin, and zeocin.

[0057] Arabinose: A simple 5-carbon sugar metabolized by E. coli. In oneembodiment, it is used as a chemical to inactivate repression and toinduce and activate expression from the promoter pBAD.

[0058] Attachment Site (att): A site specific site for recombinationthat occurs on either a phage or a chromosome. An attachment site onlambda is termed “attP”, while an attachment site of a bacterialchromosome is “attB.” Integrase mediated recombination of an attP sitewith an attB site leads to integration of the λ prophage in thebacterial chromosome.

[0059] Bacterial artificial chromosome (BAC): Bacterial artificialchromosomes (BACs) have been constructed to allow the cloning of largeDNA fragments in E. coli, as described in O'C.onner et al., Science244:1307-12, 1989; Shizuya et al., Proc. Natl. Acad. Sci. U.S.A.89:8794-7, 1992; Hosoda et al., Nucleic Acids Res. 18:3863-9, 1990; andAusubel et al., eds., Current Protocols In Molecular Biology, John Wiley& Sons (c) 1998 (hereinafter Ausubel et al., herein incorporated in itsentirety). This system is capable of stably propagating mammalian DNAover 300 kb. In one embodiment, a BAC carries the F replication andpartitioning systems that ensure low copy number and faithfulsegregation of plasmid DNA to daughter cells. Large genomic fragmentscan be cloned into F-type plasmids, making them of use in constructinggenomic libraries.

[0060] Beta: The 28 kDa lambda Beta ssDNA binding polypeptide (andnucleic acid encoding lambda beta) involved in double strand breakrepair homologous recombination. DNA encoding Beta (bet) and polypeptidechains having lambda Beta activity are also referred to herein as bet.See Examples 1 and 14 and references therein for further information.The lambda Beta protein binds to single-stranded DNA and promotesrenaturation of complementary single strand regions of DNA (see alsoKarakousis et al., J. Mol. Biol. 276:721-733, 1998; Li et al., J. Mol.Biol. 276:721-733, 1998; Passy et al., PNAS 96:4279-4284, 1999).

[0061] Functional fragments and variants of Beta include those variantsthat maintain their ability to bind to ssDNA and mediate therecombination function of lambda Beta as described herein, and in thepublications referenced herein. It is recognized that the gene encodingBeta may be considerably mutated without materially altering the ssDNAbinding function or homologous recombination function of lambda Beta.First, the genetic code is well-known to be degenerate, and thusdifferent codons encode the same amino acids. Second, even where anamino acid mutation is introduced, the mutation may be conservative andhave no material impact on the essential functions of lambda Beta. SeeStryer, Biochemistry 3rd Ed., (c) 1988. Third, part of the lambda Betapolypeptide chain may be deleted without impairing or eliminating itsssDNA binding protein function, or its recombination function. Fourth,insertions or additions may be made in the lambda Beta polypeptidechain—for example, adding epitope tags—without impairing or eliminatingits essential functions (see Ausubel et al., 1997, supra).

[0062] Biolistics: Insertion of DNA into cells using DNA-coatedmicro-projectiles. Also known as particle bombardment or microparticlebombardment. The approach is further described and defined in U.S. Pat.No. 4,945,050, which is herein incorporated by reference.

[0063] cDNA (complementary DNA): A piece of DNA lacking internal,non-coding segments (introns) and regulatory sequences that determinetranscription. cDNA may be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

[0064] Cosmid: Artificially constructed cloning vector containing thecos gene of phage lambda. Cosmids can be packaged in lambda phageparticles for infection into E. coli; this permits cloning of larger DNAfragments (up to 45 kb) than can be introduced into bacterial hosts inplasmid vectors.

[0065] Cre: The Cre recombinase is a site-specific recombinase. Itrecognizes and binds to specific sites called LoxP. Two LoxP sitesrecombine at nearly 100% efficiency in the presence of Cre, thus,permitting DNA cloned between two such sites to be efficiently removedby the Cre-mediated recombination.

[0066] De-repressible Promoter: When a repressor is bound to ade-repressible promoter transcription is substantially decreased ascompared to transcription from the de-repressible promoter in theabsence of repressor. By regulating the binding of the repressor, suchas by changing the environment, the repressor is released from thede-repressible promoter, and transcription increases. As used herein, ade-repressible promoter does not require an activator for transcription.One specific, non-limiting example is the pL promoter, which isregulated by the repressor cI, but is not activated by an activator. Thearabinose promoter is not a simple de-repressible promoter as arabinoseinactivates the repressor AraC and converts it to an activator.

[0067] In one embodiment, the de-repressible promoter is a temperaturesensitive de-repressible promoter. For example, by increasing thetemperature, the repressor is released from the promoter, or can nolonger bind to the promoter with a high affinity, and transcription isincreased from the promoter. One specific, non-limiting example is theinduction of pL promoter activity by increasing the temperature of thecell. Increased temperature inactivates the temperature-sensitiverepressor cI, allowing genes that are operably linked to the pL promoterto be expressed at increased levels. One of skill in the art can readilyidentify a repressible promoter.

[0068] In one embodiment, a de-repressible promoter is auto-regulated.One specific, non-limiting example of an auto-regulated de-repressiblepromoter is pL. If only one copy of a gene encoding cI is present, yetmany copies of the pL promoter are present, expression of cI isupregulated such that transcription is blocked from any of the pLpromoters.

[0069] Double-strand break repair recombination: A type of homologousrecombination exemplified by the lambda recombination proteins Exo, Betaand Gam, and shared by numerous other recombinase systems. A doublestrand break is the initiation point for concerted action ofrecombination proteins. Typically, an exonuclease degrades processivelyfrom the 5′ ends of these break sites, and ssDNA binding polypeptidebinds to the remaining 3′ single strand tail, protecting and preparingthe recessed DNA for homologous strand invasion (Szostak et al., Cell33:25-35, 1983; Little, J. Biol. Chem. 242:679-686, 1967; Carter et al.,J. Biol. Chem. 246:2502-2512, 1971; Lindahl et al., Science286:1897-1905, 1999). Examples of ssDNA binding polypeptides which bindto either ssDNA and/or dsDNA with 3′ overhangs and promote double-strandbreak repair recombination include lambda Beta, RecT of E. coli, Erf ofphage p22, and Rad52 in various eukaryotic cells including yeast andmammalian cells.

[0070] Electrocompetent: Cells capable of macromolecular uptake upontreatment with electroporation.

[0071] Electroporation: A method of inducing or allowing a cell to takeup macromolecules by applying electric fields to reversibly permeabilizethe cell walls. Various methods and apparatuses used are further definedand described in: U.S. Pat. Nos. 4,695,547; 4,764,473; 4,882,28;4,946,793; 4,906,576; 4,923,814; and 4,849,089, all of which are hereinincorporated by reference.

[0072] Eukaryotic cell: A cell having an organized nucleus bounded by anuclear membrane. These include lower organisms such as yeasts, slimemolds, and the like, as well as cells from multicellular organisms suchas invertebrates, vertebrates, and mammals. They include a variety oftissue types, such as, but not limited to, endothelial cell, smoothmuscle cell, epithelial cell, hepatocyte, cells of neural crest origin,tumor cell, hematopoetic cell, immunologic cell, T cell, B cell,monocyte, macrophage, dendritic cell, fibroblast, keratinocyte, neuronalcell, glial cell, adipocyte, myoblast, myocyte, chondroblast,chondrocyte, osteoblast, osteocyte, osteoclast, secretory cell,endocrine cell, oocyte, and spermatocyte. These cell types are describedin standard histology texts, such as McCormack, Introduction toHistology, (c) 1984 by J. P. Lippincott Co.; Wheater et al., eds.,Functional Histology, 2nd Ed., (c) 1987 by Churchill Livingstone;Fawcett et al., eds., Bloom and Fawcett: A Textbook of Histology, (c)1984 by William and Wilkins, all of which are incorporated by referencein their entirety. In one specific, non-limiting example, a eukaryoticcell is a stem cell, such as an embryonic stem cell.

[0073] Exo: The exonuclease of lambda (and the nucleic acid encoding the10 exonuclease protein) involved in double strand break repairhomologous recombination. See Example 1 and references therein forfurther description.

[0074] Exogenous: The term “exogenous” as used herein with reference tonucleic acid and a particular cell refers to any nucleic acid that doesnot originate from that particular cell as found in nature. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. Nucleic acid that isnaturally-occurring also can be exogenous to a particular cell. Forexample, an entire chromosome isolated from a cell of subject X is anexogenous nucleic acid with respect to a cell of subject Y once thatchromosome is introduced into Y's cell.

[0075] Extrachromosomal: Not incorporated into the chromosome orchromosomes of a cell. In the context of nucleic acids, extrachromosomalindicates an DNA oligonucleotide that is not covalently incorporatedinto the chromosome or chromosomes of a cell. Intrachromosomal refers tomaterial such as an oligonucleotide that is incorporated into thechromosome or chromosomes of a cell, such as a DNA oligonucleotidecovalently incorporated into the chromosomal DNA of a cell.

[0076] Flanking: A nucleic acid sequence located both 5′ and 3′ of anucleic acid sequence of interest. Thus, in the sequence “A—B—A”,nucleic acid sequence “A”. flanks nucleic acid sequence “B”. In onespecific, non-limiting example, nucleic acid sequence “A” is locatedimmediately adjacent to nucleic acid “B.” In another specific,non-limiting example, an linker sequence of not more than 500nucleotides is between each copy of “A” and “B,” such as a linkersequences of about 200, about 100, about 50, or about 10 nucleotides inlength. Nucleotide sequences “A” and “B” can be of any length.

[0077] Flanked nucleic acid or flanked transgene: A nucleic acidsequence flanked at a 5′- and 3′-portion by recombining sites. In oneembodiment, the nucleic acid is a transgene. In another embodiment, thenucleic acid is an antibiotic resistance cassette. In a furtherembodiment, the nucleic acid is a BAC DNA, or a gene on a BAC DNA. Inone specific, non-limiting example, the recombining site is Lox.

[0078] fLOXed nucleic acid or transgene: A nucleic acid sequence, suchas a transgene, which is flanked at a 5′- and 3′-portion by Loxrecombining sites.

[0079] Gam: A lambda protein (and nucleic acid encoding Gam) involved indouble strand break repair homologous recombination. It is believe toinhibit cellular nuclease activity such as that encoded by the recBCDand sbcC system of E. coli. See Examples 1, 7 and 14 and referencestherein for further description. Gam function, when expressed in thecell, is extremely toxic to the cell, and prevents growth. For thisreason tight controls over its expression are always required. Asdescribed herein, pL and cI 857 are able to regulate Gam expression

[0080] Functional fragments and variants of Exo and Gam: As discussedfor Beta (see “Functional fragments And Variants Of Beta”), it isrecognized that genes encoding Exo or Gam may be considerably mutatedwithout materially altering their function, because of genetic codedegeneracy, conservative amino acid substitutions, noncritical deletionsor insertions, etc. Unless the context makes otherwise clear, the termlambda Exo, Exo, or lambda exonuclease are all intended to include thenative lambda exonuclease, and all fragments and variants of lambdaexonuclease.

[0081] Gene: A nucleic acid encoding a protein product. In a specificnon-limiting example, a gene includes at least one expression controlsequence, such as a promoter, enhancer or a repressor. In anotherspecific, non-limiting example, a gene includes at least one intron andat least on exon.

[0082] Homologous arm: Nucleotides at or near 5′ or 3′ end of apolynucleotide which are identical or similar in sequence to the targetnucleic acid in a cell, and capable of mediating homologousrecombination with the target nucleic acid. Homologous arms are alsoreferred to as homology arms. In one embodiment, a homology arm includesat least 20 bases of a sequence homologous to a nucleic acid ofinterest. In another embodiment, the homology arm includes at least 30base pairs of a sequence homologous to a nucleic acid of interest. Inyet another embodiment, a homology arm includes at least 40 base pairsof a sequence homologous to a nucleic acid of interest. In a furtherembodiment, a homology arm includes from about 50 to about 100 basepairs of a sequence homologous to a nucleic acid of interest.

[0083] Homologous recombination: An exchange of homologouspolynucleotide segments anywhere along a length of two nucleic acidmolecules. In one embodiment, two homologous sequences are 100%identical. In another embodiment, two homologous sequences aresufficiently identical such that they can undergo homologousrecombination. Specific, non-limiting examples of homologous sequencesare nucleic acid sequences that are at least 95% identical, such asabout 99% identical, about 98% identical, about 97% identical, or about96% identical.

[0084] Host cell: A cell that is used in lab techniques such as DNAcloning to receive exogenous nucleic acid molecules. In one embodiment ahost cell is used to maintain or allow the reproduction of a vector, orto facilitate the manipulation of nucleic acid molecules in vitro. Ahost cell can be a prokaryotic or a eukaryotic cell.

[0085] HVJ-mediated gene transfer: A method of macromolecular transferinto cells using inactivated hemagglutinating virus of Japan andliposomes, as described in Morishita et al., J. Clin. Invest.91:2580-2585, 1993; Morishita et al., J. Clin. Invest. 94:978-984, 1994;which are herein incorporated by reference.

[0086] Inducible promoter: A promoter whose activity may be increased(or that may be de-repressed) by some change in the environment of thecell. Examples of inducible promoters abound in nature, and a broadrange of environmental or hormonal changes may activate or repress them.

[0087] Intron: An intragenic nucleic acid sequence in eukaryotes that isnot expressed in a mature RNA molecule. Introns of the presentdisclosure include full-length intron sequences, or a portion thereof,such as a part of a full-length intron sequence.

[0088] Isolated: An “isolated” biological component (such as a nucleicacid or protein) has been substantially separated or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, and proteins. Thus, nucleic acids andproteins that have been “isolated” include nucleic acids and proteinspurified by standard purification methods. The term also embracesnucleic acids and proteins prepared by recombinant expression in a hostcell as well as chemically synthesized nucleic acids.

[0089] Knockout: Inactivation of a gene such that a functional proteinproduct cannot be produced. A conditional knockout is a gene that isinactivated under specific conditions, such as a gene that isinactivated in a tissue-specific or a temporal-specific pattern. Aconditional knockout vector is a vector including a gene that can beinactivated under specific conditions. A conditional knockout transgenicanimal is a transgenic animal including a gene that can be inactivatedin a tissue-specific or a temporal-specific manner.

[0090] Linear plasmid vector: A DNA sequence (1) containing a bacterialplasmid origin of replication, (2) having a free 5′ and 3′ end, and (3)capable of circularizing and replicating as a bacterial plasmid byjoining its free 5′ and 3′ ends. Examples of linear plasmid vectorsinclude the linearized pBluescript vector and linearized pBR322 vectorsdescribed herein.

[0091] Lipofection: The process of macromolecular transfer into cellsusing liposomes. See U.S. Pat. No. 5,651,981, which is hereinincorporated by reference.

[0092] Lox: A target recombining site sequence recognized by thebacterial Cre recombinase (Cre). Specific, non-limiting examplesinclude, but are not limited to, the sequence listed as GenBankAccession No. M10494.1; LoxP (GenBank Accession No. U51223); Lox 511(Bethke and Sauer, Nuc. Acid. Res. 25:282-34, 1997); ψLOXh7q21(Thyagarajan et al., Gene 244:47-54, 2000), ψCoreh7q21 (Thyagarajan etal., Gene 244:47-54, 2000) as well as the Lox sites disclosed in Table 1of Thyagarajan et al. (Gene 244:47-54, 2000, herein incorporated byreference). In one example, LoxP sites are defined by the sequenceATAACTTCGTATAATGTATGCTATACGAAGTTAT (SEQ ID NO: 51).

[0093] A “minimal” Lox sequence is the minimal sequence recognized byCre. In one emb example, minimal Lox sequence is as described inHoekstra et al., Proc. Nat. Acad. Sci. U.S.A. 88:5457-61, 1991. Inanother example, 5′ and 3′ Lox sequences are identical.

[0094] As used herein, Lox sequences are located upstream and downstream(5′ and 3′, respectively) to a nucleic acid sequence, for example anucleic acid sequence encoding a transgene, such as a transgene encodinga therapeutic polypeptide, or a marker polypeptide.

[0095] Mini lambda: A derivative of lambda (λ) wherein most of the virallytic genes, including those required for replication and lysis, aredeleted. A mini-lambda maintains the red functions (Beta, Exo, and Gam)for homologous recombination and maintains the integration/excisionfunctions (e.g. att, integrase (int). and excisionase (xis)) to insertand excise its DNA from the chromosome.

[0096] Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer ineither single or double stranded form, including known analogs ofnatural nucleotides unless otherwise indicated.

[0097] Oligonucleotide (oligo): A single-stranded nucleic acid rangingin length from 2 to about 500 bases, for example, polynucleotides thatcontain at least 20 or 40 nucleotides (nt). Oligonucleotides are oftensynthetic but can also be produced from naturally occurringpolynucleotides.

[0098] Operably linked: A first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

[0099] Phagemid artificial chromosome: Also referred to as P1 artificialchromosome. A type of artificial chromosome allowing for stable cloningof very large DNA fragments. Phagemid artifical chromosomes are furtherdescribed in Shepherd, et al., Proc. Natl. Acad. Sci. U.S.A. 92:2629,1994; Iannou et al., Nature Genetics 6:84-89, 1994.

[0100] Phage-based recombination systems: Bacteria such as E. coliencode their own homologous recombination systems, which are used inrepair of DNA damage and to maintain a functional chromosome. Theviruses or phages that inhabit bacteria often carry their ownrecombination functions. Phage λ carries the Red recombination system.These phage systems can work with the bacterial recombination functionsor independently of them.

[0101] pL promoter: The major leftward promoter of bacteriophage lambda.Once the lambda DNA is incorporated into the bacterial chromosome,transcription from this promoter is substantially repressed by the cIrepressor. Upon inactivation of the cI repressor, for example by heatshock of a temperature sensitive mutant, transcription from the pLpromoter is activated, leading to expression of lambda genes. See FIG.1; Sambrook et al., Bacteriophage Lambda Vectors, Chapter 2 in MolecularCloning: a Laboratory Manual, 2nd Ed., (c) 1989 (hereinafter Sambrook etal.); Stryer, Control of Gene Expression in Procaryotes, Chapter 32 inBiochemistry 3rd Ed., pp. 799-823, (c) 1988 (hereinafter Stryer); andCourt and Oppenheim, pp. 251-277 in Hendrix et al. eds., Lambda II, ColdSpring Harbor Lab Press, (c) 1983 (hereinafter Court and Oppenheim).

[0102] Plasmid: Autonomously replicating, extrachromosomal DNAmolecules, distinct from the normal bacterial genome and nonessentialfor bacterial cell survival under nonselective conditions.

[0103] Polynucleotide: A double stranded or single stranded nucleic acidsequence of any length. Therefore, a polynucleotide includes moleculeswhich are 15, 50, 100, 200 nucleotides long (oligonucleotides) and alsonucleotides as long as a full length cDNA.

[0104] Unless specified otherwise, the left-hand end of single-strandedpolynucleotide sequences is the 5′ end; the left-hand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction. A nucleotide sequence 5′of a second nucleotide sequence is referred to as “upstream sequences;”a nucleotide sequence 3′ to a second nucleotide sequence is referred toas “downstream sequences.”

[0105] Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation).

[0106] Prokaryote: Cell or organism lacking a membrane-bound,structurally discrete nucleus and other subcellular compartments.

[0107] Probes and primers: A nucleic acid probe comprises an isolatednucleic acid attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, ligands, chemiluminescentagents, and enzymes. Methods for labeling and guidance in the choice oflabels appropriate for various purposes are discussed, e.g., in Sambrooket al., (1989) and Ausubel et al., (1997).

[0108] Primers are short nucleic acids, preferably DNA oligonucleotides15 nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand. The3′ hydroxyl endof the primer may be then extended along the target DNA strand throughthe use of a DNA polymerase enzyme. Primer pairs (one on either side ofthe target nucleic acid sequence) can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art.

[0109] Methods for preparing and using probes and primers are described,for example, in Sambrook et al. (1989), Ausubel et al. (1987). PCRprimer pairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as Primer (Version 0.5,© 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).Under appropriate conditions, the specificity of a particular probe orprimer increases with its length. Thus, in order to obtain greaterspecificity, probes and primers may be selected that comprise 20, 25,30, 35, 40, 50 or more consecutive nucleotides of related cDNA or genesequence.

[0110] Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as in the caseof a polymerase II type promoter, a TATA element. Enhancer and repressorelements can be located adjacent or distal to the promoter, and can belocated as much as several thousand base pairs from the start site oftranscription. Examples of promoters include, but are not limited to,the SV40 promoter, the CMV promoter, the β-actin promoter, andtissue-specific promoters. Examples of tissue-specific promotersinclude, but are not limited to: probasin (which is promotes expressionin prostate cells), an immunoglobulin promoter; a whey acidic proteinpromoter; a casein promoter; glial fibrillary acidic protein promoter;albumin promoter; β-globin promoter; an insulin promoter; and the MMTVpromoter. In yet another embodiment, a promoter is a hormone-responsivepromoter, which promotes transcription only when exposed to a hormone.Examples of hormone-responsive promoters include, but are not limitedto: probasin (which is responsive to testosterone and other androgens);MMTV promoter (which is responsive to dexamethazone, estrogen, andandrogens); and the whey acidic protein promoter and casein promoter(which are responsive to estrogen).

[0111] A hybrid promoter is a promoter that directs transcription of anucleic acid in both eukaryotic and prokaryotic cells. One specific,non-limiting example of a hybrid promoter is a PGK-EM7 promoter. Anotherspecific, non-limiting example of a hybrid promoter is PGK-Tnf.

[0112] Purified: The term purified does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedlambda Beta preparation or ssDNA binding polypeptide is one in which theprotein is more enriched than the protein is in its natural environmentwithin a cell. Preferably, a preparation of lambda Beta is purified suchthat the polypeptide represents at least 50% of the total proteincontent of the preparation.

[0113] RecA: The RecA protein is a central protein that has an activityas in the recombination function of E. coli. Homologues are found in allother organisms. RecA protein allows two homologous DNAs to find eachother among non-homologous DNAs and then trade or transfer strands witheach other. This exchange occurs by RecA binding to a single strandedregion in one of the DNAs and using that strand to search for its dsDNAhomolog, binding to the dsDNA and causing the single strand to pair withits complement in the dsDNA ultimately displacing the identical strandof the duplex. This strand transfer generates a key intermediate in theRecA-mediated recombination process.

[0114] recE recT genes and the Rac prophage: E. coli and other bacteriacontain in their chromosomes remnants of viruses. These viruses orprophages are for the most part defective and may contain only a fewgenes of the original virus. In E. coli, one defective prophage iscalled Rac. Two genes, recE and recT of the Rac prophage, encodehomologous recombination functions. These genes are normally silent butthe sbcA mutation activates their constitutive expression. Thus, thesbcA mutant is active for recombination.

[0115] Recombinases: Proteins that, when included with an exogenoustargeting polynucleotide, provide a measurable increase in therecombination frequency between two or more oligonucleotides that are atleast partially homologous. A recombinase catalyses recombination ofrecombining sites (reviewed in Kilby et al., TIG 9:413-21, 1993; Landy,Curr. Opin. Genet. Devel. 3:699-707, 1993; Argos et al., EMBO J.5:433-40, 1986). One specific, non-limiting example of a recombinase isCre. Another specific, non-limiting example of a recombinase is a Flpprotein. Other specific, non-limiting examples of a recombinase are Tn3recombinase, the recombinase of transposon gamma/delta, and therecombinase from transposon mariner.

[0116] The Cre and Flp proteins belong to the lambda/integrase family ofDNA recombinases. The Cre and Flp recombinases are similar in the typesof reactions they carry out, the structure of their target sites, andtheir mechanism of recombination (Jayaram, TIBS 19:78-82, 1994; Lee etal., J. Biol. Chem. 270:4042-52, 1995). For instance, the recombinationevent is independent of replication and exogenous energy sources such asATP, and functions on both supercoiled and linear DNA templates.

[0117] Recombinases exert their effects by promoting recombinationbetween two of their recombining sites. In the case of Cre, therecombining site is a Lox site (see U.S. Pat. No. 4,959,317), and in thecase of Flp the recombining site is a frt site. Similar sites are foundin transposon gamma/delta, TN3, and transposon mariner. Theserecombining sites are comprised of inverted palindromes separated by anasymmetric sequence (Mack et al., Nuc. Acids Res. 20:4451-5, 1992; Hoesset al., Nuc. Acids Res. 14:2287-300, 1986; Kilby et al., TIG 9:413-21,1993). Recombination between target sites arranged in parallel(so-called “direct repeats”) on the same linear DNA molecule results inexcision of the intervening DNA sequence as a circular molecule.Recombination between direct repeats on a circular DNA molecule excisesthe intervening DNA and generates two circular molecules. Both theCre/Lox and flp/frt recombination systems have been used for a widearray of purposes such as site-specific integration into plant, insect,bacterial, yeast and mammalian chromosomes (Sauer et al., Proc. Natl.Acad. Sci. U.S.A. 85:5166-70, 1988). Positive and negative strategiesfor selecting or screening recombinants have been developed (Sauer etal., J. Mol. Biol. 223:911-28, 1992). The use of the recombinant systemsor components thereof in transgenic mice, plants and insects amongothers reveals that hosts express the recombinase genes with no apparentdeleterious effects, thus confirming that the proteins are generallywell-tolerated (Orban et al., Proc. Natl. Acad. Sci. U.S.A. 89:6861-5,1992).

[0118] Recombining site: Nucleic acid sequences that include invertedpalindromes separated by an asymmetric sequence (such as a transgene) atwhich a site-specific recombination reaction can occur. In one specific,non-limiting example, a recombining site is a Lox site, such as LoxP orLox 511 (see above). In another specific non-limiting example, arecombining site is a frt site. A frt site consists of two inverted13-base-pair (bp) repeats and an 8-bp spacer that together comprise theminimal frt site, plus an additional 13-bp repeat which may augmentreactivity of the minimal substrate (e.g. see U.S. Pat. No. 5,654,182).In other, specific non-limiting examples, a recombining site is arecombining site from a Tn3, a mariner, or a gamma/delta transposon.

[0119] Selection markers or selectable markers: nucleic acid sequenceswhich upon intracellular expression are capable of conferring either apositive or negative selection marker or phenotypic characteristic forthe cell expressing the sequence. The term “selection marker” or“selectable marker” includes both positive and negative selectionmarkers. A “positive selection marker” is a nucleic acid sequence thatallows the survival of cells containing the positive selection markerunder growth conditions that kill or prevent growth of cells lacking themarker. An example of a positive selection marker is a nucleic acidsequence which promotes expression of the neomycin resistance gene, orthe kanamycin resistance gene. Cells not containing the neomycinresistance gene are selected against by application of G418, whereascells expressing the neomycin resistance gene are not harmed by G418(positive selection). A “negative selection marker” is a nucleic acidsequence that kills, prevents growth of or otherwise selects againstcells containing the negative selection marker, usually upon applicationof an appropriate exogenous agent. An example of a negative selectionmarker is a nucleic acid sequence which promotes expression of thethymidine kinase gene of herpes simplex virus (HSV-TK). Cells expressingHSV-TK are selected against by application of ganciclovir (negativeselection), whereas cells not expressing the gene are relativelyunharmed by ganciclovir. The terms are further defined, and methodsfurther explained, by U.S. Pat. No. 5,464,764, which is hereinincorporated by reference.

[0120] Selectable phenotype: A cell with a selectable phenotype is onethat expresses a positive or negative selection marker.

[0121] Sequence identity: The similarity between two nucleic acidsequences, or two amino acid sequences is expressed in terms of thesimilarity between the sequences, otherwise referred to as sequenceidentity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar are the two sequences.

[0122] Methods of alignment of sequences for comparison are well-knownin the art. Various programs and alignment algorithms are described in:Smith and Waterman, Adv. App. Math. 2:482, 1981; Needleman and Wunsch,J. Mol. Bio. 48:443, 1970; Pearson and Lipman, Methods in Molec. Biology24:307-331, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins andSharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-90, 1988; Huang et al., Computer Applications in BioSciences8:155-65,1992; and Pearson et al., Methods in Molecular Biology24:307-31,1994. Altschul et al. (Nature Genet., 6: 119-29, 1994)presents a detailed consideration of sequence alignment methods andhomology calculations.

[0123] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biological Information (NBCI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastm, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence identity using this program.

[0124] Homologues of lambda Beta, Exo and Gam, and ssDNA bindingproteins typically possess at least 60% sequence identity counted overfull-length alignment with the amino acid sequence of the protein beingevaluated (that is, lambda Beta, Exo or Gam, or ssDNA binding proteinsuch as P22 Erf, RecT, and Rad52) using the NCBI Blast 2.0, gappedblastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least 70%, at least 75%, at least80%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs will typically possess at least 75% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are described at the NCBIwebsite

[0125] One of skill in the art will appreciate that these sequenceidentity ranges are provided for guidance only; it is entirely possiblethat strongly significant homologs or other variants could be obtainedthat fall outside of the ranges provided.

[0126] Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA):ssDNA is DNA in a single polynucleotide chain; the DNA bases are notinvolved in Watson-Crick base pairing with another polynucleotide chain.dsDNA involves two or more complementary polynucleotide chains, in whichthe two polynucleotide chains are at least partially Watson-Crickbase-paired to each other. Note that a segment of DNA may be partiallyssDNA and partially dsDNA, for example if there are gaps in onepolynucleotide chain of a segment of dsDNA, or there are 5′ or 3′overhangs. ssDNA and dsDNA may contain nucleotide analogs, nonnaturallyoccurring or synthetic nucleotides, biotin, or epitope or fluorescenttags. ssDNA or dsDNA may be labeled; typical labels include radioactiveisotopes, ligands, chemiluminescent agents, and enzymes.

[0127] Target nucleic acid sequence: The nucleic acid segment which istargeted for homologous recombination. Typically, this is a segment ofchromosomal or extrachromosomal DNA in a cell. Extrachromosomal DNAharboring target nucleic acid sequences may include episomal DNA,plasmid DNA, bacterial artificial chromosome, phagemid artificialchromosomes, yeast artificial chromosomes, cosmids, and the like. Thetarget nucleic acid sequence usually harbors a gene or gene fragmentwhich will be mutated in some fashion upon homologous recombination.Examples of target nucleic acid sequences include DNA sequencessurrounding the tyr 145 UAG amber mutation of galK, as described in Yuet al., PNAS 97:5798-5983, 2000, and in Example 3 of this application;the second exon of mouse hox 1.1 gene, as described in U.S. Pat. No.5,464,764; the human hemoglobin S gene mutation as described in Example15 of this application.

[0128] Targeting frequency: The frequency with which a target nucleicacid sequence undergoes homologous recombination. For example,extrachromosomal DNA is introduced into a eukaryotic cell. Theextrachromosomal DNA has sequences capable of undergoing homologousrecombination with a target intrachromosomal DNA sequence. Afterintroducing the extrachromosomal DNA and allowing homologousrecombination to proceed, the total number of cells may be determined,and the number of cells having the target DNA sequence altered byhomologous recombination may be determined. The targeting frequency isthe number of cells having the target DNA sequence altered, divided bythe total number of cells. For example, if there are a total number ofone million cells, and 1,000 of these cells have the target DNA sequencealtered, then the targeting frequency is 1 in 1,000, or 10⁻³.

[0129] Transformed: As used herein, the term transformation encompassesall techniques by which a nucleic acid molecule might be introduced intosuch a cell, including transfection with viral vectors, transformationwith plasmid vectors, and introduction of DNA (including DNA linked toBeta protein) by electroporation, lipofection, and biolistics.

[0130] Transgene: A foreign gene that is placed into an organism byintroducing the foreign gene into embryonic stem (ES) cells, newlyfertilized eggs or early embryos. In one embodiment, a transgene is agene sequence, for example, a sequence that encodes a marker polypeptidethat can be detected using methods known to one of skill in the art. Inanother embodiment, the transgene is a conditional knockout allele.

[0131] Transgenic Animal: An animal, for example, a non-human animalsuch as, but not limited to, a mouse, that has had DNA introduced intoone or more of its cells artificially. By way of example, this iscommonly done by random integration or by targeted insertion. DNA can beintegrated in a random fashion by injecting it into the pronucleus of afertilized ovum. In this case, the DNA can integrate anywhere in thegenome, and multiple copies often integrate in a head-to-tail fashion.There is no need for homology between the injected DNA and the hostgenome. In most cases, the foreign transgene is transmitted tosubsequence generations in a Mendelian fashion (a germ-line transgenic).

[0132] Targeted insertion, the other common method of producingtransgenic animals, is accomplished by introducing the DNA intoembryonic stem (ES) cells and selecting cells in which the DNA hasundergone homologous recombination with matching genomic sequences. Forthis to occur, there is homology between the exogenous and genomic DNA,and positive selectable markers are often included. In addition,negative selectable markers can be used to select against cells thathave incorporated DNA by non-homologous recombination (randominsertion).

[0133] Upstream: Refers to nucleic acid sequences that preceed thecodons that are transcribed into a RNA of interest, or to a nucleic acidsequences 5′ of a nucleic acid of interest. Similary, “downstream”refers to nucleic acid sequences that follow codons that are transcribedinto a RNA of interest, or to nucleic acid sequences 3′ of a nucleicacid of interest.

[0134] Variants of Amino Acid and Nucleic Acid Sequences: The productionof lambda Beta, Exo or Gam, or other ssDNA binding polypeptide can beaccomplished in a variety of ways. DNA sequences which encode for theprotein, or a fragment of the protein, can be engineered such that theyallow the protein to be expressed in eukaryotic cells, bacteria,insects, and/or plants. In order to accomplish this expression, the DNAsequence can be altered and operably linked to other regulatorysequences. The final product, which contains the regulatory sequencesand the nucleic acid encoding the therapeutic protein, is operablylinked into a vector, allowing stable maintenance in a cell. This vectorcan then be introduced into the eukaryotic cells, bacteria, insect,and/or plant. Once inside the cell, the vector allows the protein to beproduced.

[0135] One of ordinary skill in the art will appreciate that the DNA canbe altered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR may be used to produce variationsin the DNA sequence which encodes lambda Beta, Exo or Gam, or otherssDNA binding proteins. Such variants may be variants that are optimizedfor codon preference in a host cell that is to be used to express theprotein, or other sequence changes that facilitate expression.

[0136] In one example, two types of cDNA sequence variants may beproduced. In the first type, the variation in the cDNA sequence is notmanifested as a change in the amino acid sequence of the encodedpolypeptide. These silent variations are simply a reflection of thedegeneracy of the genetic code. In the second type, the cDNA sequencevariation does result in a change in the amino acid sequence of theencoded protein. In such cases, the variant cDNA sequence produces avariant polypeptide sequence. In order to preserve the functional andimmunologic identity of the encoded polypeptide, such amino acidsubstitutions are ideally conservative in highly conserved regions.Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Outside of highlyconserved regions, non-conservative substitutions can more readily bemade without affecting function of the protein. Examples of conservativesubstitutions are shown in Table 1 below. TABLE 1 Original ResidueConservative Substitution Ala Ser Arg Lys Asn Gln, His Asp Glu Cys SerGln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg;Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp;Phe Val Ile; Leu

[0137] Variations in the cDNA sequence that result in amino acidchanges, whether conservative or not, should be minimized in order topreserve the functional and immunologic identity of the encoded protein.The immunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody to the protein; a variant thatis recognized by such an antibody is immunologically conserved.Particular examples of cDNA sequence variants introduce no more than 20,fewer than 10 amino acid substitutions, fewer than five amino acidsubstitutions, or about a single amino acid substitution, into theencoded polypeptide. Variant amino acid sequences may, for example, beat least 80, 90 or even 95% identical to the native amino acid sequence.

[0138] Yeast artificial chromosome (YAC): A vector used to clone DNAfragments (up to 400 kb); it is constructed from the telomeric,centromeric, and replication origin sequences needed for replication inyeast cells (see Ausubel et al.).

Use of the Lambda-encoded Red Recombination System in RecombineeringMediated by a Defective Prophage

[0139] Bacteriophage λ contains a homologous recombination system termedRed, which is functionally analogous to the RecET recombination systemof Rac. Like RecET, Red recombination requires two genes: redα or exo,which is analogous to recE, and redβ (or bet), which is analogous torecT). Exo is a 5′-3′ exonuclease that acts processively on lineardsDNA. Beta binds to the ssDNA overhangs created by Exo and stimulatesannealing to a complementary strand but cannot promote direct strandinvasion and exchange on its own. The recombination functions of Exo andBeta are again assisted by λ phage-encoded Gam, which inhibits theRecBCD activity of the host cell. λ Red-mediated recombination eventsare 10 to 1000 times more efficient than those observed in recBC sbcBCor recD strains. Because homologous recombination is increaseddramatically by the addition to the host of phage-encoded proteinfunctions, this procedure is widely applicable to any E. coli strain andto other bacterial species as well.

[0140] A defective λ prophage-based system is disclosed herein forRed-mediated recombineering (see FIG. 2). In this system, Gam, Beta, andExo are encoded by a defective lambda prophage, which is integrated intothe E. coli chromosome of a bacterial cell (e.g. E. coli) (see FIG. 6and FIG. 9). Expression of Gam, Beta, and Exo is under the tight controlof a de-repressible promoter. In the example shown, the de-repressiblepL promoter is under the control of the temperature-sensitive λ cI857repressor. At 32° C., when the repressor is active, expression of the pLpromoter and these genes is undetectable. However, when the cells areshifted to 42° C. for about a 15 minute period, the repressor isinactivated and the genes are expressed at very high levels. Incontrast, promoters that can be activated, which are present onplasmids, are notoriously difficult to control and Red and Gam functionswould be expressed even in the absence of the inducer, such asarabinose. Low-level expression of Gam causes a RecBCD defect, acondition that results in plasmid instability and loss of cellviability.

[0141] The tight regulation afforded by the prophage system, combinedwith the fact that the λ promoter, which drives Gam and Red expressionis a very strong promoter, makes it possible to achieve recombinationfrequencies that are at least 50-fold higher than those found with theplasmid-based system used previously (see Muyrers et al, Nucleic AcidsRes 27:1555-1557, 1999; Yu et al. Proc Natl Acad Sci U.S.A.97:5978-5983, 2000), and several orders of magnitude higher thanpreviously described strains in which linear recombination has beenstudied. The prophage itself is genetically stable, unlike plasmids, anddoes not rely on the presence of drug selection for maintenance.

[0142]FIG. 2 illustrates the design of primers for amplification of adsDNA recombination cassette, and a strategy for generating recombinantDNA molecules and gene replacement. The steps are outlined below.

[0143] Classical recombinant DNA technology or genetic engineering hasprimarily relied upon the presence of restriction enzyme cleavage sitesto judiciously cleave DNA and the use of DNA ligase to covalently joindifferent DNAs to make the recombinants wanted. The ability to dogenetic engineering has been simplified by the polymerase chain reaction(PCR), which allows restriction sites to be incorporated into linear PCRproducts thereby allowing more precise positioning of those sites. Allgenetic engineering technology breaks down, however, when cloningvehicles and the target contain hundreds of kilobases of DNA. Examplesinclude the bacterial chromosome and large genomic BAC clones. Even rarerestriction enzyme sites occur frequently on such large DNA moleculesmaking the effort to use unique sites impossible. Furthermore, the invitro manipulation of linear DNAs of this length is also extremelydifficult. Therefore, once large BAC cloning technology became availablein E. coli, modification of the BAC clones became the primary problem.Initially a combination of genetic engineering technology and classicalhomologous recombination techniques were adapted to modify the largegenomic clones. Classical homologous recombination in E. coli dependsupon significant (>500 bp) stretches of homology between DNAs.

[0144]FIG. 2A depicts a typical genetic engineering protocol to modify atarget on a BAC clone with a cassette and compares that technology withthe recombineering technology disclosed herein that uses special phagerecombination functions. In general, there are many steps required forclassical engineering, and the final product cannot be engineered asprecisely as by the new recombineering technology. An advance in therecombineering methodology is the use of phage recombination functionsthat generate recombination products using homologies of 50 bp (orless). Note that the target homologies in FIG. 2A and FIG. 2B arerepresented by the striped boxes. In the method outlined in FIG. 2A,those boxes must be at least 500 bp long, whereas in the method outlinedin FIG. 2B, they need only be about 40 to about 50 bp long.

[0145] Thus, in one example, genetic engineering steps to generate BACrecombinant include cleavage of the cassette DNA by a restrictionenzyme, cleavage of target on plasmid by a restriction enzyme (whereinthe vector has been pre-engineered to contain target fragments). Thecassette is joined to the plasmid by DNA ligase, and the DNA isintroduced into cells. Drug resistant (drug^(R)) clones are selected,and the plasmid is isolated. The cloned cassette is verified andsubsequently transformed into the BAC strain. Several recombinationsteps are used to introduce the cloned cassette into the BAC.

[0146] In contrast, in one non-limiting example, recombineering steps togenerate BAC recombinants can include the generation of two primers(white and black arrows, FIG. 2B), and the generation of a PCR amplifiedcassette with flanking homologies. In the example pictured in FIG. 2B,exemplary striped homology segments shown are 50 base pairs long, butthey can be about 100 base pairs in length, or from about 200 to about500 base pairs in length. Phage recombination functions are induced intoa BAC strain or BAC DNA is introduced into strain carrying recombinationfunctions. The cells containing the BAC and the recombination functionsare transformed with a PCR cassette. A recombinant is generated in vivo,and can then be detected by selection or counter-selection, by directscreening (colony hybridization), or by detecting a label on the nucleicacid (e.g. when DNA includes a DNA adduct or a marker such as biotin) Asdisclosed herein, in one specific, non-limiting example, the defective λprophage was transferred to the BAC host strain DH10B so that it can beused for BAC engineering. The modified DH10B strain called DY380 can betransformed with BAC DNA at efficiencies of 10⁻⁶ to 10⁻⁴. The utility ofDY380 cells for BAC engineering has been demonstrated by introducing a250 kbp mouse BAC that contains the neuronal-specific enolase 2 (Eno2)gene into DY380 cells by electroporation and then modifying the BAC byintroducing a Cre-expressing targeting cassette into the 3′ end of theEno2 gene using Red recombination (see Example 20). The targetingcassette was PCR-amplified from a template plasmid using chimeric 63nucleotide (nt) primers. The 3′ 21 nucleotides of each primer washomologous to the targeting cassette, while the 5′ 42 nucleotides washomologous to the last exon of Eno2 where the cassette was to betargeted (see FIG. 10). DY380 cells were then electroporated with theamplified targeting cassette and correctly targeted colonies wereobtained at an efficiency approaching 10⁻⁴ following the induction ofRed expression; no targeted colonies were obtained in un induced cells.

[0147] As also disclosed herein, the modified full length BAC waspurified and injected in mouse zygotes and a BAC transgenic lineestablished. Two other transgenic lines carrying a shorter 25 kbpsubclone of the modified Eno2 gene on pBR322 were also established ascontrols. The 25 kbp subclone carries the entire modified Eno2 codingregion as well as 10 kbp of 5′ flanking sequence and 5 kbp of 3′flanking sequence. The activity of the Cre gene in the differenttransgenic lines was then assessed by crossing the mice to ROSA26reporter mice. These mice carry a lacZ reporter that can be activated byCre recombinase. In mice carrying the full length BAC transgene, Creactivity was detected in all Eno2-positive neurons. In contrast, not allEno2-positive neurons expressed Cre in the transgenic mice carrying thesmaller 25 kbp subclone, and the pattern of Cre expression variedbetween the two different 25 kbp subclone lines. These results areconsistent with previous studies showing that regulatory sequences canbe located hundreds of kilobases from a gene, and highlight theusefulness of BAC engineering for in this case generating Cre-expressinglines for use in conditional knockout experiments.

[0148] Arabinose-inducible flpe or Cre genes have also been introducedinto the defective prophage carried in strain DY380. flpe is agenetically engineered flp that has a higher recombination frequencythan the original flp (Buchholz et al., Nat Biotechnol 16:657-662, 1998,herein incorporated by reference). The site-specific recombinases Flpeand Cre are important tools used to add or delete DNA segments (e.g.drug cassettes). Flpe and Cre expression can be induced by the additionof arabinose and used to remove the selection marker from the targetedlocus. This will be especially important in cases where the selectionmarker interferes with the expression of the targeted locus. However,even excision of the selectable marker by Flpe or Cre recombinationleaves behind the frt or LoxP site as a scar on the targeted locus.

[0149] 8Using the methods disclosed herein, conditional knockout alleles(cko alleles) can be produced. These alleles allow inactivation of agene of interest under specified biological conditions. Typically, acondition knockout (cko) allele is made by inserting recombinationsites, such as, but not limited to, LoxP sites into two introns of agene, flanking an exon, or at the opposite ends of a gene. Genes ofinterest include, but are not limited to, genes encoding polypeptidesincluding, but not limited to cytokines, hormones, structural molecules,enzymes, transcriptional factors (e.g. Evi9) and others.

[0150] Expression of a recombinase, such as, but not limited to, Cre, inmice carrying the cko allele catalyzes recombination between the LoxPsites and inactivates the gene. In one embodiment, transgenic animals,such as, but not limited to, transgenic mice (cko mice), can be producedincluding a cko allele. These mice allow a gene to be inactivated in atissue- or temporal-specific fashion. In one specific, non-limitingexample, the mice include a tissue-specific, or temporal-specificpromoter operably linked to a nucleic acid encoding a recombinase. Thus,the gene of interest is inactivated when the recombinase is expressed.

[0151] In one example of a method to produce a cko allele, two sets ofPCR primers are produced and used to amplify two homologous regions of aBAC DNA. The two homologous regions can be about 100 to about 500 basepairs in length, such as about 200 to about 500 base pairs, or about 100base pair regions. These regions of homology are used to subclone a BACof interest into a vector, such as a plasmid in a cell. In oneembodiment, a genomic fragment of about 5 to about 20 kilobases isinserted into a vector, such as a genomic fragment of about 10 to 15kilobases in length. A LoxP site is then introduced into the subclonedBAC DNA by introducing a nucleic acid sequence encoding a selectionmarker flanked by two recombination sites, such as, but not limited to,LoxP sites (e.g., a fLOXed nucleic acid encoding a selection marker),using homologous recombination.

[0152] To introduce the nucleic acid sequence encoding a selectionmarker, a vector is utilized that includes a selection marker flanked bytwo recombination sites (recombination site 1), which are in turnflanked by sequences homologous to the BAC (homology arms). The homologyarms include more than 100 base pairs homologous to the BAC DNA, such asabout 200 to about 500 base pairs that are homologous to the BAC DNA.

[0153] This vector is utilized to introduce the selection marker flankedby the two recombining sites into the BAC DNA in a host cell.Specifically, expression of Red recombination functions in a cell, suchas a bacterial cell, is used to induce recombination. In this manner,homologous recombination is used to introduce the selection markerflanked by two recombination sites (two recombination site 1) into theBAC DNA. The selectable marker can be used to identify cells that haveundergone homologous recombination.

[0154] Following homologous recombination, expression of a recombinasein the cell results in the excision of the selection marker. In onespecific, non-limiting example, the recombinase is Cre, and therecombination sites are LoxP sites. In another specific, non-limitingexample, the recombinase is Flpe, and the recombination sites are frtrecombination sites. Following expression of the recombinase, such as,but not limited to, Cre, a single recombination site (recombination site1), such as, but not limited to, a LoxP site, remains in the BAC DNA.

[0155] A second corresponding recombination site (recombination site 1,e.g. a LoxP site) is introduced at a second (e.g., a downstream) site inthe BAC DNA. In one specific, non-limiting example, the first and thesecond recombination sites are introduced into the BAC DNA such thatthey flank at least one exon included in the BAC DNA. In anotherspecific, non-limiting example, the first and the second recombinationsites are introduced into a first and a second intron of a single gene,respectively, wherein the first and the second intron are not the sameintron.

[0156] To introduce the second recombination site, a nucleic acidsequence including (1) a selectable maker flanked by a second pair ofrecombination sites (recombination site 2) is introduced into the BACDNA, and (2) a second recombination site (recombination site 1), isintroduced into the BAC DNA. In one embodiment, a vector is utilizedincluding, in 5′ to 3′ orientation, a first recombining site, a hybridpromoter operably linked to a nucleic acid encoding a selection marker,a second recombining site, and a third recombining site, wherein thefirst recombining site and the second recombining site can undergorecombination with each other in the presence of a single recombinase.

[0157] To introduce the nucleic acid sequence including the secondrecombination site the vector further includes the selection markerflanked by two recombination sites (recombination site 2) and anotherrecombination site (recombination site 1). All of these elements (e.g.,5′-recombination site 2-selection marker-recombination site2-recombination site 1-3′, or 5′-recombination site 1-recombination site2-selection marker-recombination site 2-3′ are in turn flanked byhomology arms. The homology arms include more than 100 base pairshomologous to the BAC DNA, such as about 200 to about 500 base pairsthat are homologous to the BAC DNA.

[0158] Thus, in one specific, non-limiting example, a vector isintroduced into a cell that includes:

[0159] 5′-nucleic acid homologous to the BAC DNA-recombination site2-nucleic acid encoding the selectable marker-recombination site2-recombination site 1-nucleic acid homologous to the BAC DNA-3′

[0160] Expression of Red recombination functions in a cell such as abacterial cell, can be used to induce recombination, thereby insertingthe selection marker flanked by two recombination sites (recombinationsite 2) and the additional recombination site (recombination site 1)into the BAC DNA. In one specific, non-limiting example, a nucleic acidis introduced into the BAC DNA having a configuration: 5′-recombinationsite 2-selectable marker-recombination site 2-recombination site 1-3′.In another embodiment, a nucleic acid is introduced into the BAC DNAhaving a configuration: 5′-recombination site 1-recombination site2-selectable marker-recombination site 2-3′. The selectable marker canbe used to select those cells having undergone recombination.

[0161] Recombination is then induced at recombination sites 2 using asite specific recombinase. In one specific, non-limiting example, ifrecombination sites 2 are fit recombination sites, Flpe is used toinduce recombination. In another specific, non-limiting example,recombination sites 2 are LoxP sites and Cre is used to inducerecombination. Following recombination, a recombination site(recombination site 1) remains in the BAC DNA.

[0162] In this manner, a first recombination site and a secondrecombination site (two copies of recombination site that can berecombined using a recombinase, e.g. recombination site 1) areintroduced in the BAC DNA to produce a “conditional knockout vector.”The first recombination site and the second recombination site can beintroduced flanking an exon of a gene of interest. Alternatively, thefirst recombination site and the second recombination site can beinserted each into a different exon. Upon induction of the expression ofa recombinase that specifically induces recombination at therecombination sites, a “knockout” of a gene included in the BAC DNA, asno functional protein can be produced following transcription. A diagramof this process is shown in FIGS. 17 and 18. The conditional knockoutvector can be linearized such that the BAC DNA including the gene ofinterest with the inserted recombination sites remains intact.

[0163] In one embodiment, a linearized conditional knockout vector isintroduced into embryonic stem cells. Homologous recombination can occureither upstream or downstream of the gene of interest with the insertedrecombination sites to stably integrate these nucleic acid sequencesinto a chromosome of the embryonic stem cell. The embryonic stem cellcan be used to produce a transgenic animal. Any animal can be of use inthe methods disclosed herein, including human and nonhuman animals. A“non-human animal” includes, but is not limited to, a non-human primate,a farm animal such as swine, cattle, and poultry, a sport animal or petsuch as dogs, cats, horses, hamsters, rodents, or a zoo animal such aslions, tigers, or bears. In one specific, non-limiting example, thenon-human animal is a transgenic animal, such as, but not limited to, atransgenic mouse, cow, sheep, or goat. In one specific, non-limitingexample, the transgenic animal is a mouse.

[0164] Advances in technologies for embryo micromanipulation permitintroduction of heterologous DNA into fertilized mammalian ova. Forinstance, totipotent or pluripotent stem cells, such as embryonic stemcells, can be transformed by microinjection, calcium phosphate mediatedprecipitation, liposome fusion, retroviral infection or other means. Inone embodiment, homologous recombination is induced in an embryonic stemcell, such that an exongenous DNA is integrated into a chromosome of theembryonic stem cell. The transformed cells are then introduced into theembryo, and the embryo then develops into a transgenic animal. Reviewsof standard laboratory procedures for the introduction of heterologousDNAs into mammalian (mouse, pig, rabbit, sheep, goat, cow) fertilizedova include: Hogan et al., Manipulating the Mouse Embryo, Cold SpringHarbor Press, 1986; Krimpenfort et al., Bio/Technology 9:86, 1991;Palmiter et al., Cell 41:343, 1985; Kraemer et al., Genetic Manipulationof the Early Mammalian Embryo, Cold Spring Harbor Laboratory Press,1985; Hammer et al., Nature 315:680, 1985; Purcel et al., Science244:1281, 1986; Wagner et al., U.S. Pat. No. 5,175,385; Krimpenfort etal., U.S. Pat. No. 5,175,384.

[0165] Thus, in one specific, non-limiting example, a “conditionalknockout transgenic animal” is generated including the gene of interestincluding the two recombination sites (e.g. including two copies ofrecombination site 1 in a gene of interest, such as flanking an exon ofa gene included in the BAC). To knockout expression of the gene ofinterest in the transgenic animal, a recombinase is expressed in a cellof the transgenic animal. In one specific, non-limiting example, togenerate a mouse wherein this knockout can occur, a conditional knockouttransgenic mouse can be mated to a second transgenic mouse carrying atransgene including a temporal- or tissue-specific promoter operablylinked to a transgene encoding the recombinase. Offspring are selectedthat carry the gene of interest including the two recombination sites,and the gene encoding the recombinase. In these animals, the gene isknocked out in those cells wherein the recombinase is expressed.

[0166] Selection cassettes, and vectors including these selectioncassettes, for use in these methods disclosed herein are also providedby this specification. In one embodiment, the cassette includes:

[0167] Recombinations site 2-hybrid promoter-selection marker1-recombination site 2-recomination site 1.

[0168] Suitable recombination sites include, but are not limited to,frt, LoxP, or Tn3, ISCF-1, a mariner, or a gamma/delta transposonrecombination site. In the selection cassette described above, thesequences of recombination site 2 and recombination site 1 differ fromeach other, and are recognized by different recombinases. Suitablehybrid promoters include PGK-EM7, for example, as included in PL451(ATCC Deposit No. ______, deposited Feb. 5, 2003), PL450, PL452 (ATCCDeposit No. ______, deposited Feb. 5, 2003), and PL459. Suitableselection markers include neomycin resistance, ampicillin resistance,kanamycin resistance, or any sequence that produces sensitivity orresistance to an antibiotic when introduced into a cell. Selectionmarkers further include any polypeptide sequence for which a selectionsystem is available (e.g. beta-galactosidase). An example of thisselection cassette is:

frt-hybrid promoter-selection marker-frt-LoxP

[0169] or

frt-PGK-EM7-selection marker-frt-LoxP

[0170] or

frt PGK-EM7-neo-frt LoxP

[0171] or

LoxP-hybrid promoter-selection marker LoxP-frt.

[0172] Exemplary selection cassettes of use in the methods disclosedherein are PL451 PL451 (ATCC Deposit No. ______, deposited Feb. 5, 2003)and PL452 PL451 (ATCC Deposit No. ______, deposited Feb. 5, 2003).

BAC Modification without Leaving Markers or ‘Scars’ at the Target Siteand Direct Genomic Modification

[0173] A two-step procedure for BAC targeting has been developed whereinmany kinds of mutations can be introduced into BACs without leavingbehind a selectable marker, such as a drug selection marker, at thetargeted locus. In one embodiment, a two-step procedure is utilized.This is exemplified in the following specific, non-limiting example. APCR-generated targeting cassette containing a sacB-neo fusion gene wastargeted to a BAC or other DNA. Cells containing the sacB-neo cassettetargeted to the genomic DNA of the BAC were then transformed with asecond targeting DNA to the same region. This cassette was designed toreplace the sacB-neo cassette, and in one instance, contained shortgenomic sequences that carried a more subtle mutation, such as a smallinsertion. By placing these newly transformed cells on media with 7%sucrose, selective pressure was applied against SacB expression, whichconverts sucrose to a bacteriotoxin (Muyrers et al., EMBO Rep 1:239-243,2000). Growth on sucrose plates thus selected for cells that havepotentially replaced the sacB-neo targeting cassette with the secondtargeting cassette containing the small insertion. Because spontaneousmutations occur in sacB to cause sucrose resistance at frequenciesapproaching 1 in 104, recombinants were identified among sucroseresistant colonies as those that have also become neomycin sensitive. Asdisclosed herein, by combining the power of Red recombination withselection/counterselection using sacB-neo, other kinds of geneticchanges besides insertions can also be generated, including deletionsand point mutations, and these mutations can be introduced intovirtually any large DNA molecule such as a BAC, PAC, or the E. colichromosome without any accompanying selectable marker.

[0174] The high frequency of recombination generated by the defectiveprophage system described herein also makes it possible to modify abacterial genome or a BAC in a single step without drug selection orcounterselection. In one specific, non-limiting example, a 24-bp flagtag was introduced into a 125-kbp BAC directly by recombination, withoutselection into the 5′ end of the SRY-box containing gene 4 (Sox4) (e.g.,see Examples 17 and 22). The recombinants were found by screeningindividual cells from the BAC electroporated culture.

[0175] Because homologies involved in Red-mediated recombination can bevery short, targeting cassettes can also be made by simply annealing twocomplementary synthetic ssDNA oligonucleotides together. As describedherein, a 70 bp targeting cassette constructed in this manner recombineswith the E. coli chromosome to create point mutations at frequenciesapproaching one in a thousand electroporated cells. Point mutationscorresponding to human disease-causing mutations can thus be introducedinto any human or mammalian gene carried on a BAC with ease and theaffect of this mutation on gene function assayed in a transgenic thatcarries a null mutation in the corresponding mouse gene.

Cloning DNA by Gap Repair

[0176] Fragments can be subcloned from BACs by Red-mediatedrecombination without the use of restriction enzymes or DNA ligases.Thus, any region of the BAC is amenable to subcloning, and subcloningdoes not depend on the placement of appropriate restriction enzymesites. Subcloning relies on gap repair to recombine the free ends of alinear plasmid vector with homologous sequences carried on the BAC. Anexample is shown in FIGS. 8 and 10. The linear plasmid vector with, forexample, an amp selectable marker and an origin of replication carriesthe recombinogenic ends. The vector is generated, for example, bypolymerase chain reaction (PCR) amplification using two chimericprimers. The 5′ end of each primer has homology to the extremities ofthe BAC sequence to be subcloned; the 3′ end of each primer is used toprime and amplify the linear plasmid DNA. Recombination generates acircular plasmid in which the DNA insert is retrieved from the BAC viagap repair. Circular recombinant plasmids are selected by their drugresistance (e.g. Amp^(R)) phenotype. Different sizes of fragments thatcan be subcloned depending on the cloning vector utilized. With a highcopy vector such as pBluescript, fragments up to about 25 kbp aresubcloned. However, with a lower copy vector such as pBR322 is used,fragments as large as about 80 kbp can be subcloned. These largerfragments were shown to be more accurately expressed in a tissuespecific manner (as was the entire BAC clone, see above).

A Mobilizable Lambda Prophage

[0177] As disclosed herein, recombination functions were expressed fromtheir native location in the pL operon of a lambda prophage using thenatural λ repressor controls (FIG. 9). However, one limitation of thedefective prophage system as disclosed in FIGS. 6 and 9 is that BACsunder study must be moved into recombination-proficient DY380 cellsbefore the BAC can be manipulated. In order to overcome this limitation,a novel prophage derivative has been generated that is isolated as amini-lambda circle DNA carrying a selectable marker (e.g. adrug-resistance marker such as tet^(R) cassette) and containing the exo,bet, and gam genes under control of the temperature inducible cI857repressor (FIG. 12). This mini-lambda can be transformed into anybacterial cell, such as a DH10B cell that carry a BAC. The mini-lambdathen integrates at the lambda attachment site to generate the defectiveprophage. This mobilizable prophage makes it possible to introduce theprophage into BAC-containing DH10B libraries and obviates the need totransfer the BAC to DY380 cells.

Recombineering Using ssDNA

[0178] Recombineering, or the use of a recombinase to mediaterecombination using homology arms sufficient to induce recombination, asdisclosed herein, can be performed using single-stranded oligos as thetargeting cassette). As described in the Examples below (e.g., seeExample 3), in E. coli, a single base change has been substituted in thegalK gene and a 3.3 kbp insertion removed from the galK gene usingsingle-stranded oligos. Single-stranded oligos have also been used tocure 5 different Tn10 insertions at different places on the E. colichromosome. Recombineering using single-stranded oligos is veryefficient with up to 6% of the electroporated cells being recombinant.Whereas Exo, Beta, and Gam facilitate recombination of PCR amplifieddsDNA cassettes with flanking homologies, only Beta is required forssDNA recombination (see FIG. 13). Maximum recombination is achievedwith oligonucleotides of about 70 bases in length, althougholigonucleotides of about forty to sixty bases in length can also beused to achieve recombination, albeit at a 5-fold lower frequency. Inone embodiment, ssDNA of about 40 to about 70 nucleotides in length isutilized. In another embodiment, ssDNA of about 70 to about 100nucleotides in length is utilized. In a further embodiment, a ssDNA ofabout 70 to about 1,000 nucleotides in length is utilized.Interestingly, Beta-mediated recombination activity is less efficientwhen ssDNA molecules are about 1,000 bases in length. In yet anotherembodiment, the ssDNA is labeled, such as with a biotinylatednucleotide, a methylated nucleotide, or a DNA adduct.

[0179] Recombination with either of two complementary DNAoligonucleotides has revealed that although either strand can beefficiently used for recombination, one strand is more competent forrecombination than the other. This strand bias has been examined atseveral positions around the bacterial chromosome with the result thatthe preferred strand correlates with the lagging strand of DNAreplication for each site tested. Without being bound by theory, theseresults indicated that strand bias is associated with the replicationdirection through the region being targeted and that ssDNA recombinationoccurs efficiently near the replication fork. The process of DNAreplication results in transient regions of ssDNA that may be accessibleto Beta-mediated annealing of the ssDNA oligo. Although recombinationoccurs on the leading strand, the increased recombination efficiency ofthe lagging strand oligos may reflect the increased frequency ofsingle-stranded regions during lagging versus leading strand synthesis(FIG. 13). DNA polymerase and DNA ligase could then complete the joiningof the annealed oligo to the lagging strand. Without being bound bytheory, the increased frequency of ssDNA recombination probably reflectsthe fact that ssDNA recombination occurs through a simpler mechanismthan dsDNA recombination. The ssDNA recombination may require onlyannealing of one single-stranded oligo to single-stranded regions in thereplicating target DNA. Moreover, ssDNA recombination also occurs inyeast with a strand bias that may also be dependent upon replication.The yeast functions required for this recombination are, however,unknown making the finding that only the Beta function from phage ) isrequired in E. coli that much more significant.

[0180] Using the methods disclosed herein, point mutations can beintroduced into a nucleic acid sequence of interest. In one specific,non-limiting example, a point mutation was engineered into the mouseBrca2 carried on a BAC using a 70 nt oligo. The targeting efficiency wasseveral times higher than would be found with dsDNA created by annealingoligos and at least 50 times higher than with dsDNA generated by PCR andcontaining large regions of nonhomology in their center. A 140 ntoligonucleotide has also been used to introduce a 29 amino acid in-framedeletion into exon11 of the Brca2 gene and a 1.93 kb deletion into theBAC vector backbone (Swaminathan et al. Genesis 29:14-21, 2001, hereinincorporated by reference). Finally, a 164 nt oligo has been used tointroduce a 24 bp flag tag into the 5′ end of Brca2. The targetingefficiency for the 164 nt oligo (7.7×10⁻³) was nearly the same as thetargeting efficiency for generating deletions using 140 nt oligos(8.3×10⁻³ and 5.4×10⁻³, respectively).

[0181] The disclosure is illustrated by the following non-limitingExamples.

EXAMPLES Examples 1 Modified Lambda Prophage for Defined Expression ofRecombination Proteins

[0182] The molecular genetics of lambda bacteriophage, including itslytic and lysogenic growth cycles, is described in Sambrook et al.,Bacteriophage Lambda Vectors, Chapter 2 in Molecular Cloning: aLaboratory Manual, 2nd Ed., (c) 1989 (hereinafter Sambrook et al., Ch.2); Stryer, Control of Gene Expression in Procaryotes, Chapter 32 inBiochemistry 3rd Ed., pp. 799-823, (c) 1988 (hereinafter Stryer); andCourt and Oppenheim, pp. 251-277 in Hendrix et al. eds., Lambda II, ColdSpring Harbor Lab Press, (c) 1983 (hereinafter Court and Oppenheim). Thecomplete sequence of lambda is known (see GenBank Accession No. NC001416, herein incorporated by reference).

[0183] Phage lambda has a well-characterized homologous recombinationsystem. Double strand breaks in DNA are the initiation sites for thisrecombination (Thaler et al., J. Mol. Biol. 195:75-87, 1987). Lambdaexonuclease (Exo) degrades processively from the 5′ ends of these breaksites, and lambda Beta binds to the remaining 3′ single strand tail,protecting and preparing the recessed DNA for homologous strand invasion(Little, J. Biol. Chem. 242:679-686, 1967; Carter et al., J. Biol. Chem.246:2502-2512, 1971).

[0184] The lambda recombination system containing exo and bet withoutgam is efficient at gene replacement using linear substrates withhomology arms of more than 1,000 bp in a strain lacking RecBCD nuclease(Murphy, Journal of Bacteriology 180:2063-2071, 1998). To test homologyarms of less than 100 bp long as substrates for lambda-mediatedrecombination, a lambda prophage was modified to express high levels ofphage recombination functions for a defined amount of time.

[0185]FIG. 1 depicts the defective λ prophage on the E. coli chromosome.The defective prophage contains λ genes from cI to int. The pL operon isintact and expressed under control of the temperature-sensitive lambdacI-repressor (allele cI857). A deletion (dotted line) removes the rightside of the prophage from cro through attR and including bioA (Pattersonet al., Gene 132:83-87, 1993). On the chromosome, the nadA and galoperons are to the left of the prophage, and the bio genes without bioAare to the right. Genes of the λ prophage are shown on the solid line,and genes of the host are shown on the broken line. pL and PR indicatethe early left and right promoters of λ. attL and attR indicate the leftand right attachment sites of %. The lambda genes and functions aredescribed in Sambrook et al., chapter 2, Stryer, and Court andOppenheim.

[0186] The absence of cro-repressor allows pL operon expression to befully derepressed when the temperature sensitive cI-repressor isinactivated at 42° C. The cro to bioA deletion removes the replicationand lytic genes of the prophage. The functions encoded by these lyticgenes are toxic to the cell and cause cell death within 7 minutes aftera normal prophage induction. Functions present in the pL operon are alsotoxic but kill cells only after 60 minutes of continuous induction(Greer, Virology 66:589-604, 1975; Kourilsky et al., Biochimie56:1517-1523, 1974). Thus, shifting of cells containing the pL operonconstruct from repressed conditions at 32° C. to induced conditions at42° C. allows pL operon expression. Shifting the cells back from 42° C.to 32° C. (or lower) within 60 minutes reestablishes repression andprevents cell death.

[0187] As the following examples will demonstrate, this modified lambdaprophage has produced unexpected advantages in mediating homologousrecombination. These unexpected advantages include surprisingly highrecombination efficiency, precise control of recombination functions,effective recombination with short homology arms, and ability togenerate homologous recombinants with polynucleotides other than longdouble-stranded DNA. Without wishing to be bound by a single explanationof the observed effects, it is likely that these unexpected advantagesaccrue from incorporation of the lambda red genes on the prophage intheir native context, thereby limiting the number of copies of thelambda recombination genes. In addition, use of the pL promoter confersthe ability to precisely control the timing and production of largeamounts of lambda recombination gene expression.

[0188] This system is not limited to expression in E. coli, but can alsowork in other bacteria, such as Salmonella and others. It can also workin eukaryotic cells, such as yeast or mammalian cells, with selection ofappropriate promoters, and with other modifications of the present toallow expression of the lambda recombinase genes. In one specific,non-limiting example, in another bacteria, genes between gam and N(including N but not gam) can be deleted to remove transcriptionterminators.

[0189] Although the pL promoter is used to illustrate the invention,other de-repressible, inducible or constitutive promoters could be used.Specific, non-limiting examples of inducible promoters are druginducible promoters (e.g. a tetracycline inducible promoter) metalinducible promoters (e.g. the metallathione promoter), or a hormoneinducible promoter (e.g. a steroid responsive element).

[0190] The pL promoter could also be used to drive expression of, forexample, P22 genes such as erf, or of RecE and RecT.

Example 2 Bacterial Strains, Expression of pL Operon, ElectroporationMethods, Identification of Recombinants

[0191] Bacterial strains used in this work are listed in Table 2. TABLE2 Strains Genotype WJW23 his ilv rpsl Δ(argF-lac)U169 nadA::Tn10 gal490λcl857 Δ(cro-bioA) ZH1141 W3110 Δ(argF-lac)U169 gal490 λN: lacZ Δ(N-int)cl857 Δ(cro-bioA) BR3677 lacl^(q) lacZ(M15) Δ(srl-recA)301::Tn10 DY329W3110 Δ(argF-lac)U169 nadA::Tn10 gal490 λcl857 Δ(cro-bioA) DY330 W3110Δ(argF-lac)U169 gal490 λcl857 Δ(cro-bioA) DY331 W3110 Δ(argF-lac)U169Δsrl-recA)301::Tn10 gal490 λcl857 Δ(cro-bioA) DY378 W3110 λcl857Δ(cro-bioA) W3110 “Wild-type” HME5 Δ(argF-lac)U169 λcI857 Δ(cro-bioA)HME6 Δ(argF-lac)U169 galK_(tyr145UAG) λcI857 Δ(cro-bioA) HME9Δ(argF-lac)U169 galK_(tyr145UAG) λcI857 Δ(cro-bioA) tyrTV<>cat HME10Δ(argF-lac)U169 galK_(tyr145UAG) λ cI857 Δ(cro-bioA) tyrTV<>catΔ(srl-recA)301::Tn10 HME31 Δ(argF-lac)U169 galK><catsacB λcI857Δ(cro-bioA) HME40 Δ(argF-lac)U169 INgal[galM⁺ K_(tyr145UAG)T⁺ E⁺ ]λcI857 Δ(cro-bioA) HME43 Δ(argF-lac)U169 galK_(tyr145UAG) λ(exo-int)<>cat Δ(gam-N) cI857Δ(cro-bioA) HME47 galK 34<>kan λexo<>catcI857Δ(cro-bioA) DY411 galK 34<>kan λcI857 Δ(cro-bioA) DH10B P mcrAΔ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara,leu)7649 galU galK rspL nupG DY303 DH10B [λcl857recA*] DY374 W3110gal490 nadA::Tn10 [λcl857 Δ(cro-bioA)] DY363 W3110 ΔlacU169 gal490[λcl857 (cro-bioA)<>tet^(a)] DY380 DH10B [λcl857 (cro-bioA)<>tet] EL11DH10B [λcl857 (cro-bioA)<>cat-sacB] EL250 DH10B [λcl857(cro-bioA)<>araC-P_(BAD)flpe^(b)] EL350 DH10B [λcl857(cro-bioA)<>araC-P_(BAD)cre]

[0192] Strain DY329 was constructed by transduction of ZH1141 with P1phage grown on WJW23, selecting for nadA::Tn10 tetracycline resistance(Tet^(R)) at 32° C. and then screening for the presence of a defectivelambda prophage which causes temperature sensitive cell growth at 42° C.Similar P1 transduction was used to create other strains described inTable 2 using standard media, methods, and selections (Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2^(nd) Ed., (c) 1989; Miller,Experiments in Molecular Genetics, Cold Springs Harbor Lab Press, (c)1972). The symbol < > is used to indicate a replacement generated byhomologous recombination. The symbol > < indicates an insertiongenerated by homologous recombination. A deletion at the point ofinsertion is indicated in parenthesis following the inserted gene. Theentire gal operon in HME40 is inverted (IN).

[0193] To induce expression from the pL operon and prepareelectroporation-competent cells, overnight cultures grown at 32° C. fromisolated colonies were diluted 50-fold in LB medium and were grown at32° C. with shaking to an OD₆₀₀ of about 0.4-0.8. Induction wasperformed on a 10 ml culture in a baffled conical flask (50 ml) byplacing the flask in a water bath at 42° C. with shaking (200revolutions/min) for 15 minutes. Immediately after the 15 minuteinduction, the flask was swirled in an ice water slurry to cool for 10minutes. An uninduced control culture, maintained at 32° C. throughout,was also placed into the ice slurry. The cooled 10 ml cultures werecentrifuged for 8 minutes at 5,500×g at 4° C. Each cell pellet wassuspended in 1 ml of ice-cold sterile water, transferred to a 1.5 mlplastic microcentrifuge tube, and was spun for 20 seconds at 4° C. atmaximum speed in a microcentrifuge. After washing the cell pellets asdescribed two more times, the cells were suspended in 100 μl of ice coldsterile water. This volume of competent cells is sufficient for twostandard electroporation reactions (˜10⁸ cells per reaction). Largercultures can be prepared for a greater number of reactions or forstorage of electrocompetent cells at −80° C. with 12% glycerol present.Fresh competent cells give highest efficiencies of recombination. Totransform cells by electroporation, purified linear donor DNA (1 to 10μl ) was mixed with competent cells in a final volume of 50 μl on ice,and then pipetted into a pre-cooled electroporation cuvette (0.1 cm).The amount of donor DNA used per reaction (usually 1 to 100 ng) isindicated for relevant experiments. Electroporation was performed usinga Bio-Rad Gene Pulser set at 1.8 kV, 25 μF with Pulse controller of 200ohms. Two protocols have been used interchangeably to allow segregationof recombinant from parental chromosomes within the electroporatedcells. In both protocols, the electroporated cells were immediatelydiluted with 1 ml of LB medium. In one, the cells were incubated for 1to 2 hours at 32° C. before selecting for recombinants. In the other,the cells were immediately diluted and spread on sterile nitrocelluosefilters (100 mm) on LB agar. After a 2 hour incubation at 32° C., thefilters were transferred to the appropriate agar plates required toselect for recombinants. Aliquots were also directly spread on LB agarand incubated at 32° C. to determine and examine total viable cellsafter electroporation. For drug resistant selection, each ml of LBmedium contained 10 μg of chloramphenicol, 12.5 μg of tetracycline, 20μg of kanamycin, 30 μg of ampicillin, or 50 g of spectinomycin.

[0194] Although recombinants were verified by more than one method, theprimary detection was for an altered phenotype caused by the modifiedtarget gene. Disruption or mutation of the galK gene was confirmed bythe presence of white colonies on MacConkey galactose indicator agar,disruption of the rnc gene for the endoribonuclease RNaseIII wasconfirmed by the inability of lambdoid type phage to lysogenize (Court,pp. 71-116 in Belasco et al., eds., Control of Messenger RNA Stability,(c) 1993, Academic Press, New York), and deletion of gam, kil, and cIIIin the pL operon was scored as an ability of the λ lysogen to survivegrowth at 42° C. (Court and Oppenheim; Greer, Virology 66:589-604,1975). PCR analysis was used to confirm the altered structure caused byreplacement of a gene. Southern hybridization analyses of parental andrecombinant DNAs confirmed structural changes, and DNA from therecombinant clones can be amplified by PCR and sequenced.

[0195] In addition to electroporation, any suitable method formacromolecular transfer into bacterial cells would be effective forpracticing the methods herein disclosed. For example, such methods mayinclude exposure to divalent cations, DMSO and the like as described ina variety of standard laboratory publications, such as Sambrook et al.(see particularly pages 1.74-1.84) and Ausubel et al., eds., CurrentProtocols In Molecular Biology, John Wiley & Sons (c) 1998 (hereinafterAusubel et al.), herein incorporated in their entirety.

Example 3 Homologous Recombination with Short Linear DNA Fragments

[0196] The recombination system described in Examples 1 and 2 were usedto generate a single bp mutation in the bacterial galK gene.

[0197] The galK gene encodes a galactokinase that phosphorylatesgalactose and its derivatives. The galK galactokinase phosphorylates2-deoxygalactose to generate 2-deoxygalactose phosphate (2DGP). Whileunphosphorylated 2-deoxygalactose has no impact on cell growth, 2DGP isa nonmetabolized sugar phosphate that inhibits cell growth. Thus, cellscontaining a wild type galK gene grow poorly on 2-deoxygalactose, aphenotype referred to herein as Gal+. In contrast, mutants defective ingalK grow well in the presence of 2-deoxygalactose (Dog), and have aphenotype referred to herein as DogR-(Adhya, pages 1503-1512 inEscherechia coli and Salmonella typhimurium: Cellular and MolecularBiology, Neihardt et al. eds., American Society of Microbiology, 1987).The DogR-phenotype enables ready selection of cells harboring successfulrecombination events.

[0198] Complementary 70 base pair oligonucleotides were synthesized andannealed to each other. The annealed DNA was homologous to an internalcoding segment of the bacterial galK gene, except that a UAU codon(TYR-145) was changed to a UAG amber codon. A homologous recombinationevent between this 70 base pair DNA fragment and the galK geneintroduces a premature stop codon in the galK gene, and is referred toherein as the galK amber mutation. This mutation produces a truncatedgalK gene product that lacks function.

[0199] The 70-bp DNA fragment was transferred by electroporation intogalK+cells (HME5) that had been induced for lambda pL operon expressionby growth at 42° C. for 15 minutes. After electroporation with themutant DNA (100 ng) or mock electroporation without DNA, the cells werespread on minimal 0.4% glycerol agar medium with 0.2% 2-deoxygalactosepresent. Spontaneous resistant mutants occurred frequently (10⁻⁴) in theabsence of mutant DNA. Despite this, the addition of the mutant DNAenhanced the frequency of resistant mutants dramatically, generating onemutant per 500 electroporated cells.

[0200] To determine that temperature induction was required, anotherbatch of cells that had not been induced for recombination function wastested in the same way. In this treatment, no discernable effect ofadded mutant DNA was observed. This indicated that both induction of pLoperon expression and mutant DNA addition were required for the enhancedsurvival. Without wishing to be bound by a single explanation of theobserved effects, it is believed that the expressed lambda functionsallowed for efficient recombination of this short linear mutant DNA withthe chromosomal galK gene.

[0201] Colonies surviving 2-deoxygalactose treatment were screened fortheir Gal phenotype on indicator plates, and all tested had theGal−phenotype expected for a galK amber mutant. To test specificallywhether the galK amber mutation was present, four independentGal−colonies were tested by transducing cultures of each with alambdaimm21 phage that carries the tRNA_(tyr) suppressor allele supF.The four mutants tested were suppressed to a Gal+phenotype. Finally, thepresence of the amber mutation in galK was verified by PCR amplificationand sequence analysis of the galK gene segment from the chromosome.

[0202] This example demonstrates that controlled expression of lambdarecombination genes from a defective lambda prophage promotessurprisingly efficient homologous recombination in bacterial cells, evenwith short linear segments of DNA having very short homology arms.

[0203] Importantly, the high recombination frequency indicates thatrecombinants were identified without the need to apply positive ornegative selection methods. DNA hybridization probes are thus designedto detect point mutations, deletions, insertions or other modificationsof cellular DNA. Standard colony hybridization or in situ hybridizationtechniques can be used to detect cells in which recombination hasoccurred. Alternatively, enrichment methods for mutation detection areused, particularly for detecting point mutations. Such enrichmentmethods are described in Gocke et al., Annals of the New York Academy ofSciences 906:31-38, 2000, which is herein incorporated by reference inits entirety. One example of a suitable enrichment is the mismatchamplification mutation assay described by Cha et al., PCR Applicationsand Methods 2:14-20, 1992, herein incorporated by reference in itsentirety.

[0204] The fact that the DNA fragments with short homology arms are ableto recombine in vivo opens a vast array of new possibilities forgenerating recombinant DNA. Several steps normally involved ingenerating recombinant DNA molecules are eliminated. Restriction enzymedigests are not required to generate DNA fragments, and DNA ligasereactions are not required to join different DNA fragments at noveljunctions. The cell generates the completed recombinant precisely joinedthrough homologous recombination.

[0205] The efficiency of recombination approaches 0.1% of survivingcells from a standard electroporation. At this efficiency, unselectedcolonies could be screened for recombinant DNA using colonyhybridization techniques, eliminating the need for selection steps.Thus, this recombination protocol makes the bacterial chromosome andplasmid DNA amenable to almost any type of desired change. This includesdirected mutagenesis of a gene, a gene segment, or even a base.

Example 4 Preparation of Linear DNA Cassettes Greater than 1000 bp inLength

[0206] Standard Polymerase Chain Reaction (PCR) conditions were used toamplify linear DNA fragments with the Expand™ High Fidelity PCR systemof Boehringer Mannheim. The chloramphenicol resistant (Cm^(R)) cassettecat was amplified from pPCR-Script Cam (Stratagene) with primers5′TGTGACGGAAGATCACTTCG (SEQ ID NO: 1) and 5′ACCAGCAATAGACATAAGCG (SEQ IDNO: 2). The tetracycline resistant (Tet^(R)) cassette tet was amplifiedfrom Tn10 with primers 5′CTCTTGGGTTATCAAGAGGG (SEQ ID NO: 3) and5′ACTCGACATCTTGGTTACCG (SEQ ID NO: 4). The ampicillin resistant (AP^(R))cassette amp was amplified from pBluescript (Stratagene) with primers5′CATTCAAATATGTATCCGCTC (SEQ ID NO: 5) and 5′AGAGTTGGTAGCTCTTGATC (SEQID NO: 6). The kanamycin resistant cassette kan was amplified from Tn5with primers 5′TATGGACAGCAAGCGAACCG (SEQ ID NO: 7) and5′TCAGAAGAACTCGTCAAGAAG (SEQ ID NO: 8). PCR products were purified usingQiagen PCR purification kits and concentrated if necessary by ethanolprecipitation. The amplified linear DNAs were suspended in sterile wateror TE buffer (10 mM Tris-Cl pH7.5; 1 mM EDTA) and quantified byspectroscopy. DNA in water was stored at −20° C. The inventors avoidedPCR product purification schemes from gels in which the DNA is subjectto ultraviolet irradiation.

[0207] In order to design primers for amplification of a recombinationcassette, recombinant oligonucleotides were chemically synthesized withthe 5′ 30 to 50 nucleotides identical to sequences at the target nucleicacid sequence, and with the 3′ 20 nucleotides homologous to the ends ofthe cassette to be introduced. A cassette is generated by PCR that isflanked by the 30 to 50 base homologies present at the target.

[0208] Cells carrying the target DNA either on the chromosome or on aplasmid are induced for Exo, Beta and Gam function. These cells are madecompetent for electroporation and mixed with the amplified cassette.Following electroporation, recombination occurs between the homologoussequences on the linear cassette and the target replaces the targetsegment with the cassette.

[0209] The 50 nt galK homology segments (rectangles) used for theexperiment described in Table 3 are:5′GTTTGCGCGCAGTCAGCGATATCCATTTTCGCGAATCCGGAGTGTAAGAA (SEQ ID NO: 9) and5″TTCATATTGTTCAGCGACAGCTTGCTGTACGGCAGGCACCAGCTCTTCCG (SEQ ID NO: 10)

[0210] In one embodiment, the cassette is a drug resistance marker butcan be any DNA if the target sequence in the subsequent steps can becounter-selected. The transcription of the marker cassette has beenoriented arbitrarily in the same direction as the target region beingreplaced. The primers contain two parts: a 5′ end homologous to flankingregions of the target DNA, and a 3′ end that primes the cassette DNA forreplication. The PCR using these primers and a DNA template containingthe marker cassette generates a linear DNA product with the cassetteflanked by target homology. Note that if transformation with thetemplate DNA will generate the selected phenotype (for example, thetemplate is a plasmid), the template is then eliminated. Plasmidtemplate DNA can be destroyed by treatment with DpnI following the PCR;DpnI cuts methylated GATC template DNA leaving the newly replicatedunmethylated DNA intact. Once a linear cassette has been generated, itcan be stored and used as the template for subsequent PCRs.

Example 5 Gene Replacement by Targeted Homologous Recombination

[0211] Having demonstrated in Example 3 that 70-bp linear DNA can directmutations to a specific target, a synthetic DNA having 50-bp galK DNAsegments flanking the cat (chloramphenicol resistance, or Cm^(R))cassette was constructed for targeting a galK gene replacement by cat.

[0212] The linear cat cassette with flanking galK DNA was made by PCRusing chemically synthesized primers, as described in Example 4.

[0213] Data from these experiments are presented in Table 3. DY330competent cells were electroporated with 100 ng of the cat cassettetargeted to replace either galK (galK< >cat) or prophage genes cIII kilgam (cIII kil gam< >cat; see FIG. 1 for map of prophage genes). Totalrecombinants per electroporation are shown in the rightmost column, “CmRrecombinants.” The cat cassette was transferred by electroporation intogalK+cells which had either been heat-induced for pL operon expression(15 minute temperature shift to 42° C., as indicated by “15” in thecenter column of Table 3) or not induced (maintained at 32° C.;indicated by “0” in the center column of Table 3). See also Example 2for description of induction and other methods. After electroporation,Cm^(R) recombinants were selected at 32° C., and then quantified.

[0214] As shown in Table 3, Cm^(R) colonies were only found in theheat-induced culture. All 50 Cm^(R) colonies tested had a Gal−phenotypeon MacConkey galactose indicator agar, indicating the presence of thegalK< >cat replacement. The symbol < > indicates a replacement generatedby homologous recombination techniques, for example, galK< >catindicates that the bacterial galK gene is replaced by cat usinghomologous recombination techniques. TABLE 3 Target Site* 42° C., minCm^(R) Recombinants GalK 0 <1 GalK 15 2.5 × 10⁴ CIII kil gam 0 <1 CIIIkil gam 15 5.0 × 10⁴

[0215] In similar experiments using the same 50-bp homologous arms, thegalK has been exchanged for kan, amp, and tet cassettes by selecting forKm^(R), AmP^(R), and Tet^(R), creating galK< >kan, galK< >amp, andgalK< >tet replacements, respectively.

[0216] To test whether this approach also works at other positions onthe bacterial chromosome, a linear cat cassette was created flanked by50-bp DNA segments found immediately upstream and downstream of the rncgene encoding RNaseIII. The rnc gene is thought to be non-essential(Takiff et al., Journal of Bacteriology 171:2581-2590, 1989); therefore,it was tested whether an exact substitution of the cat coding region forthe rnc coding region (from AUG to codon 224) could be made using therecombination techniques herein described. In this construct, cat istranscribed from the rnc promoter, and the 5′ primer used to generatecat started at the cat initiation codon.

[0217] Following procedures used for galK as described in Examples 2 and3, Cm^(R) colonies were found, but only in the induced culture. TheCm^(R) colonies tested had a Rnc mutant phenotype, as described inExample 2.

[0218] Two other rnc< >cat recombinants were made. One replaced sequencefrom the AUG start to codon 126 of rnc, and the other from the AUG startto codon 192 of rnc. These two recombinants generate cat::rnc genefusions with an rnc mutant phenotype. Different sets of primers werechosen to detect unambiguously the wild type and/or recombinant alleles.This PCR procedure follows guidelines set forth by yeast researchers incharacterizing chromosomal replacements in yeast (Winzeler et al.,Science 285: 901-906, 1989). The PCR analysis of the recombinantsverified the loss of the rnc+gene and the predicted structures of thethree rnc< >cat gene replacements.

[0219] In this example, several different genes on the bacterialchromosome and on plasmids have been substituted with drug resistancemarkers. However, it is also possible to create recombinants in whichthe desired product does not include a selectable marker. Genes havebeen fused to cassettes encoding specific tags such as the greenfluorescent protein. Fusion tags can be placed precisely in the gene tobe modified, for example, by any of the following strategies. In one,the unselected cassette is joined to a selectable drug marker, and bothare recombined into the chosen location selecting for drug resistance.In another, the cassette is recombined into its location by substitutingit for a negative selection marker like sacB (Bloomfield et al., MolMicrobiol 5:1447-57, 1991). This strategy permits cloning of any DNA. Ina third strategy, the recombinants are screened non-selectively by DNAhybridization with probes specific to the cassette.

[0220] In these experiments, the desired recombination product wasusually obtained. However, some recombination products were unexpected.In two cases, an attempt was made to knockout essential genes, andsurprisingly it was possible to select a few rare recombinants. Theseturned out to be diploid for the region of the targeted gene, since theycarried the wild type and the mutant copy of the gene as determined byPCR analysis. Rare diploid regions of the bacterial chromosome are knownto occur spontaneously in growing cells at a frequency of about 0.1%(Haack and Roth, Genetics 14:1245-1252, 1995). Because an essential genewas targeted, these rare diploids were selected. This was only possiblebecause the recombination is so efficient.

[0221] This example demonstrates that the methods of this invention canbe used to promote efficient gene replacement by homologousrecombination. The gene replacements were made throughout the bacterialchromosome. In Example 7, it is shown that the methods can be used tomodify extrachromosomal nucleic acids, such as plasmids, bacterialartificial chromosomes, cosmids, phagemids, and the like.

Example 6 Induction Time, DNA Amount, and Homology Arm Length AffectTargeting Efficiency

[0222] Induction time. FIG. 3 shows the effect of induction time onrecombination. The strain DY330 was grown at 32° C. to OD₆₀₀=0.4 to 0.8,heat-induced at 42° C. for the times indicated and then madeelectrocompetent (see Example 2 for description of methods). A linearcat cassette (10 ng) was used to target the cIII kil gam genes of theprophage. Total Cm^(R) recombinants were plotted versus the time ofinduction.

[0223] Induction of pL operon expression for only five minutes enhancedrecombination activity. FIG. 3 reveals that by 7.5 minutes of heatinduction a maximum efficiency is reached. This maximum level ismaintained for induction times from 7.5 to 17.5 minutes with somereduction occurring for times longer than 17.5 minutes. Expression ofthe pL operon for longer than 60 minutes causes cell death.

[0224] Cells harboring the defective lambda prophage may be grown attemperatures other than 32° C. In general, it is undesirable to growcells at temperatures >37° C., because such temperatures lead to partialinactivation of the cI repressor, and leaky expression from the pLpromoter. In general, it is also undesirable to grow cells attemperatures below 20° C., because of slow growth. One skilled in theart would also recognize that there is a considerable degree of latitudewith regard to time and temperature of induction. For example,expression of lambda recombination genes from the pL operon could beinduced at temperatures as low as 38° C., generally allowing for longertimes of induction. The limit for protein expression in E. coli is about45° C.

[0225] Donor DNA amount. FIG. 4 shows the effect of amount of the linearDNA cassette on recombination. The strain DY330 was grown at 32° C. toOD₆₀₀=0.4 to 0.8, induced at 42° C. for 15 minutes and then madeelectrocompetent. Different amounts (1, 10, 100, 300, 1,000 ng) of alinear cat cassette (1 kbp in length) were used to target the cIII kilgam genes of the prophage. Total Cm^(R) recombinants were plotted versusthe DNA amount at 42° C.

[0226]FIG. 4 shows that targeting efficiency increased in a near linearrelationship with increasing concentration of donor DNA in the rangefrom 10⁸ (1 ng) to 10¹⁰ (100 ng) molecules per electroporation. Asaturating level of linear DNA is reached at 3×10 molecules yielding7.5×10⁴ recombinants per 2×10⁸ cells electroporated. Thus, the methodsof this invention may be practiced over a broad range of oligonucleotideconcentrations.

[0227] Homology length. FIG. 5 shows the effect of homologous arm lengthon recombination. The strains DY330 (recA +)(filled circles) and DY331(recA−)(open circles) were grown at 32° C., induced at 42° C. for 15minutes and then made electrocompetent. A linear cat cassette (100 ng)was used to target the cIII kil gam genes of the prophage. Thehomologous arm length of the cassette was varied from 0 to 1,000 bp. Theprimers containing the 0 to 50 bp homologies were chemically synthesizedas described (FIG. 2). The cassette containing 1,000 bp homologous armswas made by PCR using primers 1,000 bp away on each side of an existing(cIII kil gam)< >cat disruption in the cell. Total Cm^(R) recombinantswere plotted versus the homologous arm length.

[0228] Several pairs of primers were made to amplify the cat cassettefor targeting the chromosome and designed each pair with a differentlength of flanking homology. The length of the homology segment on theprimers varied by increments of 10 bases from 10 to 50 bases. A nestedset of linear cat cassettes was made with the primers. Another linearcat cassette was constructed flanked by 1,000 bp of homology. This setof linear DNAs was then tested for recombinational targeting efficiencyas shown in FIG. 5. No recombinants were found with 10 bp of homologyand less than ten recombinants were found in each of three experimentswith 20 bp of homology. From 20 bp to 40 bp of homology, homologousrecombination increased by four orders of magnitude. From 40 bp to 1,000bp of homology, recombination increased 10-fold.

[0229] These data indicate that the methods herein disclosed may bepracticed with surprisingly short homology arms, as few as 20-40residues. However, homology arms of 30 or greater residues increaseefficiency.

Example 7 Gene Replacement on Plasmids and BACs: in vivo Cloning

[0230] To determine if this method could be used to modify plasmid DNA,the procedures described in Examples 2-3 were followed to modify plasmidpGB2, a derivative of pSC101 (Bemardi et al., Nucleic Acids Res.12:9415-9426, 1984). A cat cassette was synthesized in vitro andrecombined in vivo with pGB2 to replace the spectinomycin resistancegene with cat conferring Cm^(R) on the cell carrying the recombinantplasmid.

[0231] The same experiment, performed on pBR322 derivatives, generatedrecombinants, but they were joined in tandem to non-recombinant plasmidsas dimers and higher multimers. Induction of Gam expression from ourprophage inactivates RecBCD nuclease. In the absence of RecBCD, pBR322derivatives replicate by a rolling circle mode (Feiss et al, Gene17:123-130, 1982), and the plasmid converts from monomers to multimers.This is specific for pBR322-type replicons as the pGB2 type did not formmultimers.

[0232] To generate simple recombinants of pBR322 derivatives, theprotocol was modified by coelectroporating the recA* strain DY331 withcircular plasmid DNA (0.1 ng) and a linear drug cassette. Recombinantplasmid monomers were readily selected and isolated.

[0233] In addition to plasmids, the method is also suitable fortargeting genes on bacterial artificial chromosomes, phagemid artificialchromosomes, yeast artificial chromosomes, cosmids, and the like.Homologous recombination between target nucleic acid sequences on BACsand synthetic oligonucleotides (such as ds DNA fragments or PCRfragments) is carried out in bacterial cells bearing the defectivelambda prophage shown in FIG. 1 and described in Example 1. Syntheticoligonucleotides (such as short annealed dsDNA fragments or PCRfragments) are electroporated into bacterial cells as described inExample 2. Analysis for successful recombination events is by selectivePCR amplification using specific primers for the introduced sequence, orby selective amplification approaches such as the mismatch amplificationmutation assay described by Cha et al., PCR Applications and Methods2:14-20, 1992, or by direct hybridization using specific probes. Usingthis approach recombination frequencies of up to 1:500 have beenobserved, regardless of the strand targeted. Thus, this system isextremely useful in manipulation of and rapid screening for recombinantsin BAC vectors. The unexpectedly high efficiencies eliminate the needfor introducing selectable markers, or modifying such markers on theBAC.

Example 8 Requirement of RecA for Targeted Recombination

[0234] The requirement for RecA were tested in targeted homologousrecombination by repeating the experiment described in FIG. 5 but usinga recA⁻ strain. Surprisingly, recombination efficiency was depressedonly about 10-fold in the recA− mutant for the arm lengths tested (FIG.5). Thus, RecA function is not required, and recA− strains mediateefficient homologous recombination with linear DNA fragments havinghomology arms of 30 bp or greater.

[0235] This result was unexpected. The λ recombination system is knownto function in cells lacking the bacterial RecA function (Brooks andClark, J. Virol. 1:283-293, 1967). However, the recombination in recAmutants under conditions used by others is reduced more than 50-foldrelative to levels in recA⁺ cells (Stahl et al., Genetics 77:395-408,1974; Murphy, J. Bacteriol. 180:2063-2071, 1998).

Example 9 Lambda Genes Promote Targeted Recombination of dsDNA in WildType E. coli

[0236] To determine which lambda genes promote targeted recombination ofdsDNA, a set of replacement deletions were generated in the pL operon ofthe prophage using cat and amp cassettes. In the center column of Table4, the parentheses indicate the deletions made by the recombinationevent within the prophage (refer to FIG. 1 for a linear map of theprophage genes). Each of these newly made deletions was verifiedstructurally by PCR analysis and tested for targeted recombination of atet gene cassette into galK.

[0237] Electrocompetent cells from strains indicated in Table 4 wereheat-induced for 15 minutes at 42° C., and electroporated with 10 ng oflinear galK< >tet. The results are presented as total number of Tet^(R)recombinants per electroporation in the right hand column of Table 4.TABLE 4 Strain Prophage Recombinant DY330 wild-type 4,100 DY392(hin-int)<>amp 2,000 DY351 (sieB-kil)<>cat 4,400 DY386 (hin-int)<>amp1,650 (sieB-kil)<>cat DY349 (gam)<>cat 0 DY360 (bet)<>cat 0 DY359(exo)<>cat 0

[0238] Table 4 shows that only exo, bet, and gam deletions affectedgalK< >tet targeted recombination. Deletion of any one of these threegenes eliminated the recombination, whereas deletion of all other genesin the pL operon had little if any effect.

[0239] To show that the gam< >cat substitution was not polar on bet andexo, the gam gene was expressed in trans and shown to complement thedefect. Thus, although the entire pL operon was used in studiesdescribed here, only exo bet and gam functions are needed forrecombination with double-stranded DNA cassettes >100-200 bp made byPCR.

Example 10 Efficient Recombination with Single-Strand DNA

[0240] To evaluate recombination between exogenous single-strand DNA andthe E. coli chromosome, the experiments described in this Example wereperformed. In addition, the experiments also address the role of thebacterial recA recombination genes in mediating the observed effects.

[0241] Expression from the pL operon was heat-induced, cells were madeelectroporation-competent, and 70-mer oligonucleotides wereelectroporated into cells, all as described in Example 2. Strains HME9and HME10 both harbor the galK amber mutation, and are therefore Gal− inphenotype. They differ from each other in that HME9 is recA+, whereasHME10 is recA−. Thus in this experiment recombination efficiencies inrecA+ and recA−cells are compared.

[0242] The 70-mer single-stranded oligonucleotides used in thisexperiment were designed to restore wild type galK gene activity(hereinafter galK+) upon successful recombination, thereby producing aGal+phenotype (i.e., ability to grow on minimal media with galactose asthe sole carbon source). The 70-mer corresponding to the transcriptionalnon-template DNA strand of galK was (SEQ ID NO: 11)5′AAGTCGCGGTCGGAACCGTATTGCAGCAGCTTTATCATCTGCCGCTGGACGGCGCACAAATCGCGCTTAA.

[0243] 70-mer single-stranded DNA of either SEQ ID NO: 11 or itscomplement was electroporated into cells. Alternatively, the two 70-merswere first annealed to each other and then electroporated into cells asdouble strand DNA. A successful recombination event was identified byrestoration of the Gal+phenotype. Table 5 indicates the number of galK+recombinants per viable cell, ×10⁻⁴.

[0244] Table 5 presents the number of recombinants observed ×10⁻⁴ afterelectroporation of the HME9 or HME10 strains with DNA in the indicatedforms. Efficient recombination was observed with double strand DNA,similar to that previously described in Examples 3 and 5. Surprisingly,single-strand DNA was about equally or even more efficient thandouble-stranded DNA in producing homologous recombination, regardless ofstrand used. In this experiment, recombination efficiencies for thedouble-stranded DNA was about 1 in 3800 cells (2.6-2.7×10⁻⁴), whereasrecombination efficiency for single-stranded counterclockwise DNA was 3-to 7-fold higher. In other strains (e.g. DY374) recombination of singlestrand linear DNA as frequent as one nadA⁺ recombinant per 20 viablecells has been observed. In addition, efficient recombination wasobserved in both recA+and recA−cells, establishing that therecombination events did not require the bacterial recA gene products.TABLE 5 /DNA used in electroporation dsDNA cc-ssDNA cw-ssDNA Strain Used(about 1 μg) (about 0.7 μg) (about 0.6 μg) HME9 2.6 18.5 2.2galK^(am)λCI857Δ(cro-bio) HME10 2.7 9 0.35 galK^(am)λCI857Δ(cro-bio)recA⁻

Example 11 Efficient Generation of Large Deletions by Recombination withSingle-strand DNA

[0245] Example 10 demonstrates efficient lambda-mediated recombinationusing ssDNA to generate a single base change in the E. coli chromosome.This example demonstrates similar high efficiency when the approach isused to generate large deletions.

[0246] Using the methods described in Examples 2, 3, and 10, lambdaoperon expression was heat-induced, cells were renderedelectroporation-competent, and 70-mer single-stranded DNA of either SEQID NO: 11 or its complement was electroporated into cells. The DNA waselectroporated into two strains: one containing the galK amber mutation,and one in which the galK gene was interrupted by a cat-sacB cassetteprecisely inserted at the position of the amber mutation in galK. Thestrains were otherwise genetically identical. A successful recombinationevent was identified by restoration of the Gal+phenotype. Table 6indicates the number of galK+recombinants per viable cell, ×10⁻⁴.

[0247] The data in Table 6 demonstrate that recombination efficiency issimilar using the same oligonucleotide, regardless of whetherlambda-mediated ssDNA recombination is being used to generate a singlebase change or a large deletion, removal of the 3264 bp cat-sacBcassette. In both cases, the method is highly efficient in generatingrecombinants. TABLE 6 DNA used in electroporation cc-ssDNA cw-ssDNAStrain Used (200 ng) (200 ng) HME6 15 3 galK^(am)λCI857Δ(cro-bio) HME3110 0.5 galK<>catsacB λCI857Δ(cro-bio)

[0248] The length that is reasonable to synthesize chemically limits thelength of single strand oligonucleotides used for recombination. It isdemonstrated herein that two oligonucleotides that have a complementaryoverlap region at their 3′ ends, when co-electroporated into DY411cells, can anneal and generate recombinants with chromosomal orextrachromosomal DNA (FIG. 14). This recombination requires theinduction of the pL operon and the Gam, Beta, and Exo functions. A galKmutation in which the kan cassette was placed in galK in a way to delete34 bp of the galK gene was created by recombineering. Two oligos weresynthesized that were 70 bases long, with 34 bases of the deleted regionat their 3′ ends, that were complementary and can act to anneal the twooligos together. The 5′ end of each oligo contained 36 bases of homologyto each side of the 34 bp deletion caused by kan. Each oligo alonecannot generate gal+ recombinants but mixed together they generated upto 10⁵ recombinants per 10⁸ cells electroporated. Oligos with the samesequence but shortened from their 3′ end to overlap by only 2 bases didnot yield recombinants. However, overlaps of 10 bases or more generatedrecombinants. Preannealing was not required and the two oligos can bemixed and used directly for electroporation.

[0249] If the ends of the overlaps are filled in by DNA polymerase a 104bp duplex is generated. This dsDNA generates only slightly morerecombinants then the DNA with 10 to 34 base overhangs. Thus, multipleoverlapping (by >10 bases) oligonucleotides of any even number can beused to yeild recombinants, in which the most outside oligonucleotideshave 5′ overlaps. The end oligonucleotides also have 30-50 bases ofhomology to the targeted region. The use of multiple overlappingoligonucleotides allows production of long recombination substrateswithout use of PCR. The central oligonucleotide(s) can be any cassetteenvisioned to be used for dsDNA recombination. This recombination withoverlapping oligonucleotides having outside 5′ overhangs is mostefficient with Exo, Beta, and Gam, but can be recombined by Beta alone(without Exo and Gam) in the cell. This greatly simplifies therecombination procedure (as only Beta is required). Although the 104 bpduplex DNA recombines more efficiently if Exo, Beta and Gam are present,recombination also occurs in the absence of Gam, albeit at a lowerefficiency (the duplex requires both Exo and Beta for recobination).

[0250] Similar overlapping synthetic oligonucleotides can be generatedwith 3′ overhangs of 34 bases that can be coelectroporated into cells.These are also recombined into targets defined by homology at the ends.Again, only Beta is required for this recombination. In this case, Exois not required, and further Exo does not stimulate recombination. Inone embodiment, multiple oligonucleotides can be overlapped as above tospan longer distance. As long as the outermost oligonucleotides have 3′overhangs, recombination will be Exo independent. The efficiencies ofthe present system allows the detection of recombinants in this case.

[0251] Examples 10 and 11, taken together, document that the methodsdisclosed herein can be practiced with ssDNA oligonucleotides. Thissurprising result enables high efficiency homologous recombination withsynthetic DNA of single or double strandedness.

[0252] The present system allows the limit of synthetic oligonucleotidesize to be increased dramatically by overlapping oligonucleotides. Inaddition, the system allows recombination of these DNAs to be carriedout with Beta alone, or Exo with Beta but without Gam. Recombinationwithout the requirement for Gam is important because Gam is the toxicfunction that was a limiting factor in the previously described methods.As the present system requires only Beta, a constitutive promoter can beutilized.

Example 12 Effect of ssDNA Length on Recombination Efficiency

[0253] In Examples 10 and 11, lambda-mediated recombination was used toefficiently incorporate 70-mer ssDNAs into the E. coli chromosome. Inthis example, the effect of oligonucleotide length on recombinationefficiency was investigated.

[0254] Using the methods described in Examples 2, 3, 5, 10 and 11,lambda operon expression was heat-induced, cells were renderedelectroporation-competent, and ssDNA oligonucleotides (200 ng each) wereelectroporated into E. coli HME9 strain cells. The electroporated ssDNAoligonucleotides included the 70-mer of SEQ ID NO: 11, a 60-merconstructed by removing the last 5 nucleotides from both the 5′ and 3′ends of SEQ ID NO: 11, and a 50-mer, 40-mer, 30-mer, or 20-merconstructed by removing the last 10, 15, 20, or 25 nucleotides,respectively, from both the 5′ and 3′ ends of SEQ ID NO: 11. As inexample 10, the ssDNA oligonucleotides used in this experiment were alldesigned to restore the galK+gene upon successful recombination, therebyconferring upon the cell a Gal+phenotype. Table 7 indicates the numberof galK+recombinants per viable cell, ×10⁻⁴. TABLE 7 Oligonucleotidelength 0 20 30 40 50 60 70 Efficiency 0.004 0.01 0.47 4 4 6 22 (×10⁻⁴)

[0255] As the data in Table 7 demonstrate, recombination efficiencyincreases with increasing ssDNA length. Recombination efficiency was lowwhen the ssDNA used was a 20-mer, but increased considerably with a30-mer. Efficiency was near optimal with a 40-mer, and increased to 1 in450 viable cells with a 70-mer. Hence, specific examples of theinvention use single-stranded DNA molecules at least about 40nucleotides in length.

[0256] Without wishing to be bound by a single explanation of theobserved effects, the inventors currently believe that observedlength-efficiency relationship may reflect published data indicatingthat lambda Beta protein binds stably to DNA sequences of 36 bases orlonger, but does not bind as well to shorter oligonucleotides (Mythiliet al,, Gene 182:81-87, 1996).

Example 13 Lambda Beta Protein Mediates Efficient Recombination withssDNA

[0257] To determine whether lambda Beta protein was sufficient tomediate recombination between exogenous ssDNA and the E. colichromosome, the efficiency of recombination was investigated in a strainthat expressed lambda Beta, but not Exo or Gam.

[0258] For these experiments, the HME43 strain was used. Its genotype isidentical to the HME6 strain, except that the lambda prophage containsadditional genetic deletions, from int through exo and from gam throughN (see FIG. 1). In addition, the cat gene conferring the Cm^(R)phenotype is inserted between attL and bet.

[0259] Using the methods described in Examples 2, 3, and 10-12,expression of the modified lambda operon was heat-induced, cells wererendered electroporation-competent, and 70-mer ssDNA of SEQ ID NO: 11(200 ng) was electroporated into cells. Using this procedure, the HME43strain expresses lambda Beta protein, but does not express gam, exo, orany other prophage encoded genes. A successful recombination event wasidentified by restoration of the Gal+phenotype. Table 8 indicates thenumber of galK+recombinants per viable cell, ×10⁻⁴ TABLE 8 RecombinationEfficiency Strain Prophage Modifications (× 10⁻⁴) HME43 (int-exo)<>cat,(gam-N)<>Δ 7.7

[0260] In contrast to Example 9 using PCR-generated double-stranded DNA,the data presented in this example establish that lambda Beta alone issufficient to mediate efficient recombination between ssDNA and the E.coli chromosome. Moreover, two or more overlapping oligonucleotides maybe used, if they have a 3′ overhang and more than about 10 bp ofoverlap. Overlapping oligonucleotides with a 5′ overhang also promotehomologous recombination with Beta alone. However, for 5′ overhangs, exoand gam (or a similar exonuclease and RecBCD-inhibition function) appearto enhance maximal efficiency.

[0261] A modification of the method is to place DNA encoding other ssDNAbinding polypeptides under control of the pL promoter. For example, thestrain HME43 is further modified to delete bet and insert DNA encodingP22 Erf, RecT, or Rad52. Expression of the ssDNA binding polypeptide isinduced by temperature shift as it is for induction of lambda betexpression. Exo and Gam, or proteins with similar function, can also beplaced under control of the pL promoter. Moreover, other inducible orconstituitive promoters can be used.

Example 14 Ex vivo Combination of ssDNA With Lambda Beta MediatesEfficient Homologous Recombination

[0262] Single-strand DNA can be combined with lambda Beta protein priorto electroporation into cells, and mediated efficient recombinationbetween the ssDNA and the host DNA.

[0263] Lambda Beta proteins may be prepared by techniques known in theart (Karakousis et al., J. Mol. Biol. 276:721-731, 1998), andpreincubated at 37° C. with single-strand oligonucleotides of 20-mer orgreater length. In this example, ssDNA oligonucleotides of SEQ ID NO:11, a 60-mer constructed by removing the last 5 nucleotides from boththe 5′ and 3′ ends of SEQ ID NO: 11, and a 50-mer constructed byremoving the last 10 nucleotides from both the 5′ and 3′ ends of SEQ IDNO: 11 were used. Typically, lambda Beta protein concentration is about2.5 μM and DNA concentration about 5 μM, but the method is effectivewith a broad range of protein and DNA concentrations (for example, from0.1 μM to 10 mM protein, and 0.01 μM to 10 mM ssDNA). Alternatively, theBeta protein and ssDNA can be coelectroporated into cells withoutpremixture or preincubation.

[0264] The DNA and protein is electoporated into E. coli using methodsdescribed in Examples 2 and 3. In this example HME 43 strain is used,but numerous other strains are suitable. Expression of the modifiedlambda operon is one set of cells is heat-induced, and a second set ofcells is maintained at 32° C. Both sets of cells are renderedelectroporation-competent, and 70-mer ssDNA of SEQ ID NO: 11 (200 ng) iselectroporated into both heat-induced and uninduced cells. Using thisprocedure, the HME43 strain expresses lambda Beta protein upontemperature shift to 42° C., but does not express Beta from bet or anyother prophage-encoded genes in the absence of a temperature shift. Asuccessful recombination event is identified by restoration of the Gal+phenotype.

[0265] In this experiment, high efficiency recombination is observed inboth heat-induced and uninduced cells. Moreover, it is believed thatapproximately equally high efficiency recombination is observed whenthese techniques are followed in E. coli strains that contain no lambdaprophage genes.

[0266] This approach can be modified by substituting other ssDNA bindingpolypeptides for lambda Beta, such as p22 Erf, RecT and Rad52. Thetarget nucleic acid sequence may be on the bacterial chromosome, or onexogenous DNA such as a bacterial artificial chromosome, phagemidartificial chromosome, plasmid, cosmid, or the like. Moreover, there isno particular requirement for a specific bacterial species; thesesingle-strand DNA binding polypeptides will mediate efficientrecombination in a broad range of bacteria. Indeed, these polypeptideswill mediate efficient recombination in eukaryotic cells as well, as inExample 15.

Example 15 Lambda Beta Protein Mediates Efficient HomologousRecombination in Eukaryotic Cells

[0267] The ex vivo approach described in Example 14 may be used totarget genes in eukaryotic cells for homologous recombination. Ineukaryotic cells, transfection of the ssDNA with lambda Beta protein maybe accomplished by electroporation as in Examples 2, 3 and 14, or by themethods of Chang et al., Biochimica et Biophysica Acta, 153-160, 1992,Keating and Toneguzzo, Bone Marrow Purging and Processing, 491-498,1990, or other electroporation protocols known in the art. In addition,a variety of means for macromolecular transfer methods are known to theart, including calcium phosphate-DNA co-precipitation (Ausubel et al.),DEAE-dextran-mediated transfection (Matthews et al., ExperimentalHematology 21:697-702, 1993) polybrene-mediated transfection (Costelloet al., Gene Therapy 7:596-604, 2000), microinjection (Davis et al.,Blood 95:437-44, 2000), liposome fusion and lipofection (Veit et al.,Cardiovascular Research 43:808-22, 1999), protoplast fusion (Schaffner,Proc. Natl. Acad. Sci. U.S.A. 77:2163, 1980), inactivatedadenovirus-mediated transfer (Wagner et al., Proc Natl Acad Sci U.S.A.89:6099-6103, 1992), hemagglutin virus of Japan-(HVJ)-mediated transfer(Morishita et al., Journal of Clinical Investigation 93:1458-1464,1994), biolistics (particle bombardment) and the like. Any suchmacromolecular transfer approach is suitable. Design of dsDNA moleculesfor facilitating homologous recombination with eukaryotic genes is wellknown in the art (for example, as described in Mansour, Nature336:348-352, 1988; Shesely, PNAS 88:4294-4298, 1991; Capecchi, M. R.,Trends in Genetics 5:70-76, 1989; U.S. Pat. No. 6,063,630).

[0268] Cells to be transfected with exogenous DNA are combined with aDNA construct comprising the exogenous DNA, targeting DNA sequences and,optionally, DNA encoding one or more selectable markers. The resultingcombination is treated in such a manner that the DNA construct entersthe cells. This is accomplished by subjecting the combination toelectroporation, microinjection, or other method of introducing DNA intovertebrate cells. Once in the cell, the exogenous ssDNA is integratedinto cellular DNA by homologous recombination between DNA sequences inthe DNA construct and DNA sequences in the cellular DNA.

[0269] For example, the target nucleic acid is the beta-globin gene inhematopoietic stem cells from a patient with sickle cell anemia(Beutler, Disorders of Hemoglobin, Ch. 107 in Harrison's Principles ofInternal Medicine, 14^(th) ed. © 1998, herein incorporated byreference). The sickle cell Beta globin gene harbors a point mutationthat substitutes a Val for Glu at position six of the polypeptide chain,resulting in an abnormal hemoglobin which is prone to inappropriatepolymerization. The methods of this invention can be used to correct themutation.

[0270] Hematopoetic stem cells from a sickle cell patient are isolated,cultured, and expanded ex vivo as is known in the art (Brugger, Seminarsin Hematology 37[1 Suppl 2]:42-49, 2000; Dao et al., Blood 92:4612-21,1998; Aglietta et al., Haematologica 83:824-48, 1998; Emerson, Blood87:3082-8, 1996). A 60-mer ssDNA oligonucleotide of SEQ ID NO: 12(AACAGACACC ATGGTGCACC TGACTCCTGA GGAGAAGTCT GCCGTTACTG CCCTGTGGGG) issynthesized and partially purified by standard techniques (Pfleiderer etal., Acta Biochimica Polonica 43:37-44, 1996; Anderson et al., AppliedBiochemistry & Biotechnology 54:19-42, 1995, herein incorporated byreference).

[0271] After culture and ex vivo expansion, about 10⁶ hematopoetic stemcells are suspended in 0.4 mL PBS containing 0.1% glucose, about 10 μMpurified lambda Beta protein, and about 1 μg ssDNA oligonucleotide ofSEQ ID NO: 12. The cell suspension is electroporated in a 1-mL cuvetteat 280V and 250 μF with a Gene Pulser (Bio-Rad Laboratories Inc.,Hercules, Calif., USA). Cells are then plated and cultured. Homologousrecombinants harboring the mutation are identified and clonallyisolated, further expanded ex vivo, and may be returned to the patient,or cultured for additional in vitro study.

[0272] Those skilled in the art will recognize that a broad range ofssDNA and ssDNA binding polypeptide concentrations will be effective, asin Example 14. For example, both ssDNA and ssDNA binding protein may bepresent in concentrations ranging from 0.001 μM to 100 mM; or from 0.1mM to 1 μM; or from 1 μM to 100 μM. Oligonucleotide length can be variedin accordance with parameters presented in Example 12. There is noparticular upper limit on oligonucleotide length. In addition, two ormore oligonucleotides can be included which have complementary 5′ ends,thereby creating 3′ overhangs which are effective substrates for ssDNAbinding polypeptides such as lambda Beta. In addition, RecT, P22 Erf,Rad52, and other double strand break repair ssDNA binding polypeptidesmay be substituted for lambda Beta. Culture and electroporationconditions are readily variable without materially reducing homologousrecombination. Moreover, nucleic acid may be introduced into the cell byany suitable macromolecular transfer method.

[0273] Other types of stem cells can be used to correct the specificgene defects associated with cells derived from such stem cells. Suchother stem cells include epithelial, liver, lung, muscle, endothelial,mesenchymal, neural and bone stem cells.

[0274] Alternatively, certain disease states can be treated by modifyingthe genome of cells in a way that does not correct a genetic defect perse but provides for the supplementation of the gene product of adefective gene. For example, endothelial cells can be used as targetsfor human gene therapy to treat disorders affecting factors normallypresent in the systemic circulation. In model studies using both dogsand pigs endothelial cells have been shown to form primary cultures, tobe transformable with DNA in culture, and to be capable of expressing atransgene upon re-implantation in arterial grafts into the host organism(Wilson et al., Science 244:1344, 1989; Nabel et al., Science 244:1342,1989). Since endothelial cells form an integral part of the graft, suchtransformed cells can be used to produce proteins to be secreted intothe circulatory system and thus serve as therapeutic agents in thetreatment of genetic disorders affecting circulating factors. Examplesof such diseases include insulin-deficient diabetes, alpha-1-antitrypsindeficiency, and hemophilia. Epithelial cells, myocytes and hepatocytesare also useful cell types for therapeutic production of proteins.

[0275] The method is also useful for knockout or modification of genesin embryonic stem (ES) cells. Such cells have been manipulated tointroduce transgenes. ES cells are obtained from pre-implantationembryos cultured in vitro (Evans et al., Nature 292:154-156, 1981;Bradley et al., Nature 309:255-258, 1984; Gossler et al., Proc. Natl.Acad. Sci. U.S.A. 83:9065-9069, 1986; Robertson et al., Nature322:445-448, 1986; U.S. Pat. No. 5,464,764). Oligonucleotides designedto target specific gene segments in the ES cell are combined with lambdaBeta protein or other ssDNA binding polypeptide and introduced into EScells by electroporation or other transformation methods. Theoligonucleotides may be designed as a series of overlapping segmentswith 3′ overhangs. Such transformed ES cells can thereafter be combinedwith blastocysts from a non-human animal. The ES cells thereaftercolonize the embryo and can contribute to the germ line of the resultingchimeric animal (Jaenisch, Science 240:1468-1474, 1988).

[0276] For example, sequences encoding positive selection markerneomycin resistance gene are synthesized as a series of overlapping70-mer oligonucleotides, 20 base pairs of overlap and 3′ overhangs. The3′ terminal oligonucleotides are designed to insert into the second exonof the mouse hox 1.1 gene as described in U.S. Pat. No. 5,464,764.Because the overlapping oligonucleotides combine to encode apromoterless neomycin resistance gene, only those that successfullyincorporate into the targeted mouse hox 1.1 second exon will express theneo gene product and have the neomycin resistance phenotype. Thetargeting is designed to provide the synthetic neomycin resistance genewith an operable promoter and translation start derived from the mousehox 1.1 gene. The targeting DNA is also designed so that randomincorporations elsewhere in the ES cell genome are unlikely to beoperably linked to any promoter to allow transcription and translation.

[0277] The series of overlapping oligonucleotides with 3′ overhangs(about 200 nanograms each) are combined with 10 μM lambda Beta proteinand introduced into ES cells by electroporation using the PromegaBiotech X-Cell 2000. Rapidly growing cells are trypsinized, washed inDMEM, counted and resuspended in buffer containing 20 mM HEPES (pH 7.0),137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM dextrose, and 0.1 mMbeta-mercaptoethanol. Just prior to electroporation, theoligonucleotides and lambda Beta protein are added to 10⁷ ES cells ineach 1 ml-cuvette. Cells and DNA are exposed to two sequential 625 V/cmpulses at room temperature, allowed to remain in the buffer for 10minutes, then plated in non-selective media onto feeder cells.

[0278] Following two days of non-selective growth, the cells aretrypsinized and replated onto G418 (250 μg/ml) media. Thepositive-selection is applied for three days. Because of the highefficiency of lambda Beta-mediated recombination, the need for furtherselection (for example, negative selection by introducing a thymidinekinase gene and selecting with ganciclovir) can be obviated.Appropriately transformed, G418-resistant cells are grown innon-selective media for 2-5 days prior to injection into blastocysts(according to the method of Bradley in: Teratocarcinomas and EmbryonicStem Cells, A Practical Approach, edited by E. J. Robertson, IRL Press,Oxford (1987), p. 125).

[0279] Blastocysts containing the targeted ES cells are implanted intopseudo-pregnant females and allowed to develop to term. Chimericoffspring are identified by coat-color markers and those males showingchimerism were selected for breeding offspring. Those offspring whichcarry the mutant allele can be identified by coat color, and thepresence of the mutant allele reaffirmed by DNA analysis by tail-blot,DNA analysis.

[0280] Thus, the method markedly simplifies the construction of geneknockouts and gene modifications in ES cells. In addition to itspossible relevance to plant, animal and human gene therapy, the methodwill simplify the construction of transgenic animals harboring eithergene knockouts or gene modifications.

[0281] As described for stem cells and in Example 14, a broad range ofssDNA and ssDNA binding polypeptide concentrations will be effective.For example, both ssDNA and ssDNA binding protein may be present inamounts ranging from 0.001 μM to 100 mM; or from 0.1 μM to 1 μM; or from1 μM to 100 μM. Oligonucleotide length can be varied in accordance withparameters presented in Example 12. There is no particular upper limiton oligonucleotide length. In addition, two or more oligonucleotides canbe included which have complementary 5′ ends (for example with 10, 20,30, 40 bp complementary 5′ ends), thereby creating 3′ overhangs whichare effective substrates for ssDNA binding polypeptides such as lambdaBeta. In addition, RecT, P22 Erf, Rad52, and other double strand breakrepair ssDNA binding polypeptides can be substituted for lambda Beta, inthe same ranges described for lambda Beta. Those skilled in the art willrecognize that culture and electroporation conditions are readilyvariable without materially reducing homologous recombination. Moreover,nucleic acid may be introduced into the cell by any suitablemacromolecular transfer method.

Example 16 Homologous Recombination in Plants

[0282] The methods disclosed herein are also applicable to themanipulation of plant cells and ultimately the genome of the entireplant. A wide variety of transgenic plants have been reported, includingherbaceous dicots, woody dicots and monocots. For a summary, see Gasseret al., Science 244:1293-1299 (1989). A number of different genetransfer techniques have been developed for producing such transgenicplants and transformed plant cells. One technique used Agrobacteriumtumefaciens as a gene transfer system (Rogers et al., Methods Enzymol.118, 627-640, 1986). A closely related transformation utilizes thebacterium Agrobacterium rhizogenes. In each of these systems a Ti or Riplant transformation vector can be constructed containing border regionswhich define the DNA sequence to be inserted into the plant genome.These systems previously have been used to randomly integrate exogenousDNA to plant genomes.

[0283] Preferably, DNA designed for homologous recombination with atarget DNA sequence in plants are combined with lambda Beta protein orother ssDNA protein and directly transferred to plant protoplasts by wayof methods analogous to that previously used to introduce transgenesinto protoplasts. Concentration of the DNA and ssDNA binding proteinsare as described in Example 15 (see, e.g. Paszkowski et al., EMBO J.,3:2717-2722, 1984; Hain et al., Mol. Gen. Genet., 199, 161-168, 1985;Shillito et al. Bio./Technology 3:1099-1103, 1985; and Negrutiu et al.,Plant Mol. Bio. 8:363-373, 1987). Alternatively, the PNS vector iscontained within a liposome which can be fused to a plant protoplast(see, e.g. Deshayes et al., EMBO J. 4:2731-2738, 1985) or is directlyinserted to plant protoplast by way of intranuclear microinjection (see,e.g. Crossway et al., Mol. Gen Genet. 202:179-185, 1986, and Reich etal., Bio/Technology 4:1001-1004, 1986). Microinjection can be used fortransfecting protoplasts. The DNA and ssDNA binding proteins can also bemicroinjected into meristematic inflorescences. De la Pena et al.,Nature 325:274-276, 1987. Finally, tissue explants can be transfected byway of a high velocity microprojectile coated with the DNA and ssDNAbinding proteins analogous to the methods used for insertion oftransgenes (see, e.g. Vasil, Bio/Technology 6:397, 1988; Klein et al.,Nature 327:70, 1987; Klein et al., Proc. Natl. Acad. Sci. U.S.A.85:8502, 1988; McCabe et al., Bio/Technology 6:923, 1988; and Klein etal., Genetic Engineering, Vol 11, J. K. Setlow editor (Academic Press,N.Y., 1989)). Such transformed explants can be used to regenerate forexample various serial crops. Vasil, Bio/Technology 6:397, 1988.

[0284] Once the DNA and ssDNA binding protein have been inserted intothe plant cell by any of the foregoing methods, homologous recombinationtargets the oligonucleotide to the appropriate site in the plant genome.As in previous examples, the oligonucleotide may be a series ofoverlapping ssDNAs with 5′ or 3′ overhangs. Depending upon themethodology used to transfect, selection is performed on tissue culturesof the transformed protoplast or plant cell. In some instances, cellsamenable to tissue culture may be excised from a transformed planteither from the F0 or a subsequent generation.

[0285] The amino acid composition of various storage proteins in wheatand corn, for example, which are known to be deficient in lysine andtryptophan may also be modified. PNS vectors can be readily designed toalter specific codons within such storage proteins to encode lysineand/or tryptophan thereby increasing the nutritional value of suchcrops. For example, the zein protein in corn (Pederson et al., Cell29:1015, 1982) can be modified to have a higher content of lysine andtryptophan by the vectors and methods disclosed herein.

Example 17 Materials and Methods Used in Examples 18-22

[0286] Bacterial strains. All of the strains used except DH10B weremaintained at 32° C. because of the temperature inducible prophage.DY303 was constructed by infecting DH10B cells (Gibco) with a λ phagecarrying recA (λcI857 recA⁺) (a gift from F. W. Stahl) and lysogens wereselected. Strain EL11 was constructed by replacing the tet gene of DY380with a cassette containing the cat and sacB genes by selecting CmR. EL11cells are Tet S, Cm^(R) and sensitive to 2% sucrose. Strain EL250 wasconstructed by replacing the cat-sacB cassette of EL11 cells with araCand the arabinose promoter-driven flpe recombinase gene (P_(BAD)flpe)selecting in the presence of sucrose. EL250 cells are resistant to 2%sucrose. Strain EL350 was constructed in a similar manner except for Creinstead of flpe.

[0287] Construction of plasmids. The IRES-eGFPcre-FRT-kan-FRT targetingcassette was PCR amplified from pICGN21, which was constructed bysubcloning a 1.9 kbp HindIII/AccI-digested and filled-in FRT-kan-FRTfragment from pFRTneo into the NotI/BclI-digested and filled-in cloningsite of pIRESeGC. The FRTneo was constructed by amplifying the kan genealong with the Beta lactamase promotor from pEGFP-C1 (Clontech) withprimers 5′CTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCGTCAGGTGGC ACTTTCGGG (SEQID NO: 13) and 5′CTCAGAAGAACTCGTCAAGAAGG (SEQ ID NO: 14). The amplifiedfragment was then targeted between the frt sites in pNeoβ-gal(Stratagene). The pIRESeGC was generated by inserting the 2 kbpNheI/MluI-digested and filled-in eGFPcre fragment from pEGC into the 3.5kbp BamHI-digested and filled-in cloning site of pNTRlacZPGKneoloxP(Arango et al., Cell 99: 409-19, 1999). The pEGC was generated bysubcloning a 1.05 kbp EcoRI/KpnI PCR fragment containing the Cre genefrom pGKmncre into the EcoRI/KpnI site of pEGFP-C1. This PCR fragmentwas generated by amplifying the Cre gene from pGKmncre with primers5′GTAGGTACCTCGAGAATCGCCATCTTCCAGCAGGC (SEQ ID NO: 15) and5′TCGAATTTTCTGCATCCAATTTACTGACCGTACACC (SEQ ID NO: 16), which containEcoRI and KpnI cleavage sites, respectively, at their 5′ ends.

[0288] To construct the pTamp vector, the amp-targeted pBeloBAC11 wasfirst generated by replacing the LoxP site in pBeloBAC11 (Shizuya etal., Proc. Natl. Acad. Sci. 89: 8794-7, 1992) with the PCR amplified ampgene from PEGFP (Clontech). The primers used for amplification are5′GCAAGTGTGTCGCTGTCGACGAGCTCGCGAGCTCGGACATGAGGTTGTCTTA GACGTCAGGTGGCAC(SEQ ID NO: 17) and5′CATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGAACCTCAC GTTAAGGGATTTTGGTC(SEQ ID NO: 18), which are homologous to the amp gene of pEGFP (inplain) and to sequences flanking the LoxP site in pBeloBAC11 (initalic). A 2.4-kbp PCR fragment amplified from amp-targeted pBeloBac11with primers 5′GCAGGATCCAGTTTGCTCCTGGAGCGACA (SEQ ID NO: 19) and5′TGCAGGTCGACTCTAGAGGATC (SEQ ID NO: 20) was then cloned into theXhoI/XbaI and filled-in site of pCS (Stratagene) to create the pTampvector. The 2.4 kbp amp cassette containing an amp gene along with 920bp of 5′, and 370 bp of 3′, pBel0BAC11 vector sequence flanking the LoxPsite can be released by BamH1 digestion and used directly to replace theLoxP site in any pBeloBAC11-derived BACs with amp.

[0289] The pKO4 vector containing the cat-sacB targeting cassette is aderivative of pKO3 (Link et al., J. Bacteriol. 179: 6228-37, 1997) inwhich 605 bp had been deleted between cat and sacB.

[0290] The araC-P_(BAD)flpe targeting cassette was amplified frompBADflpe, which was constructed by subcloning a 1.4 kb PstI/KpnIfragment from pOGFlpe (Buchholz et al., Nat. Biotechnol. 16: 657-62,1998) into pBAD/MycHis-A (Invitrogen). The araC-P_(BAD)cre targetingcassette was amplified from pBADcre, which was constructed byintroducing a 1.2 kb HindIII/NcoI fragment from pGKmncre into pBAD/His-C(Invitrogene).

[0291] Amplification primers for targeting or GAP repair cassette DNAs.For all primers listed below, nucleotides in italics are homologous tothe targeted sequence, while those in plain text are homologous toamplification cassettes. The Tet^(R) cassette used for targeting cro-bioin DY330 was amplified from Tn10 with primers:

[0292] 5′TGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATGCTCTTGGGTTATC AAGAGGG(SEQ ID NO: 21) and

[0293] 5′GGCGCTGCAAAAATTCTTTGTCGAACAGGGTGTCTGGATCACTCGACATCTTG GTTACCG(SEQ ID NO: 22). The cat-sacB cassette used for replacing the tet genein DY363 was amplified from pKO4 with primers:

[0294] 5′TGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATGCTGTGACGGAAG ATCACTTCG(SEQ ID NO: 23) and

[0295] 5′GGCGCTGCAAAAATTCTTTGTCGAACAGGGTGTCTGGATCCTGAGGTTCTTAT GGCTCTTG(SEQ ID NO: 24). The araC-P_(BAD)flpe and araC-P_(BAD)cre cassettes usedfor replacing the cat-sacB in EL11 were amplified from pBADflpe andpBADcre with primers:

[0296] 5′TGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATGAAGCGGCATGCA TAATGTGC(SEQ ID NO: 25) and

[0297] 5′GGCGCTGCAAAAATTCTTTGTCGAACAGGGTGTCTGGATCCTGTGTCCTACTCAGGAGAGCGTTC (SEQ ID NO: 26). The IRES-eGFPcre-FRT-kan -FRT cassetteused for targeting the Eno2 locus was amplified from pIGCN21 withprimers:

[0298] 5′CGCTTCGCGGGACATAATTTCCGAAATCCCAGTGTGCTGTGAGCCAAGCTATCGAATTCCGCC (SEQ ID NO: 27) and

[0299] 5′GAGGCTCCAGGAGAATGAGATGTTCCCGCGTTCAGGCAAGCGCTATTCCAGAAGTAGTGAGGA (SEQ ID NO: 28). The oligonucleotides used to target theflag cassette into the 5′ end of the Sox4 gene were annealed andpolymerase-extended using primers: (SEQ ID NO: 29)5′GCGAGCGTGTGAGCGCGCGTGGGCGCCCGGCAAGCCGGGGCCATGGATTTACAAGGATGACGACGATAAGGTACAACAGA and (SEQ ID NO: 30)5′GGCCAGCAGAGCCTCAGTGTTCTCCGCGTTGTTGGTCTGTTGTACCTTATCGTCGTCATCCTTGTAATCCATGGCCCCC.

[0300] The linear pBR322 derivative used to subclone the 25-kbp fragmentfrom the modified Eno2 locus was amplified with primers:

[0301] 5′CTCTCCATGCCTGTCTGGGTGAGGGTGGCCCAGGGGCGATGGCTATGAGAGAGGTCGACTTCTTAGACGTCAGGTGGCAC (SEQ ID NO: 31) (Eno2-C-L1) andGCAATGCAGAGAAGCCTTGTACTGGGATGACAGAGACGGAGGGGAAGAGGAGGCGGCCGCGATACGCGAGCGAACGTGA (SEQ ID NO: 32) (Eno2-C-R1/2). Theamplification primers for the other experiments were: 48-kbp modifiedfragment,

[0302] 5′GACTTCTATGACCTGTACGGAGGGGAGAAGTTTGCGACGTGACAGAGCTGGTCGTCGACTTCTTAGACGTCAGGTGGCAC (SEQ ID NO: 33) (Eno2-C-L2/3/4) andEno2-C-R1/2; 60-kbp modified fragment, Eno2-C-L2/3/4 and

[0303] 5′GCCCCATACACGTAAATGTACATAGAATCACACAGCATCACTTCTATGGATGCGGCGGCCGCGATACGCGAGCGAACGTGA (SEQ ID NO: 34) (Eno2-C-R3); 80-kbp modifiedfragment, Eno2-C-L2/3/4 and

[0304] 5′CATCCAGTAGAACTTGGGAGTGAAGCTAGAGCCAAGGCCATCTAAGTGACAGGCGGCCGCGATACGCGAGCGAACGTGA (SEQ ID NO: 35) (Eno2-C-R4). These primerscontained 5′ regions homologous to the target sequence and 3′ regionshomologous to pBR322. PCR products were purified using a Qiaex II gelextraction kit (Qiagen) and digested with DpnI to remove contaminatedtemplate.

[0305] Preparation of electrocompetent cells and generation ofrecombinants. For BAC modification, overnight cultures containing theBAC were grown from single colonies and were diluted 50-fold in LBmedium and grown to an OD₆₀₀=0.5-0.7. 10-ml cultures were then inducedfor Beta, Exo, and Gam expression by shifting the cells to 42° C. for 15minutes followed by chilling on ice for 20 minutes. Cells then werecentrifuged for 5 minutes at 5,500 g at 4° C. and washed with 1.5 ml ofice-cold sterile water three times. Cells were then resuspended in 50 ,lof ice-cold sterile water and electroporated. For BAC transformation,the induction step was omitted.

[0306] Cell transformation was performed by electroporation of 100-300ng linear DNA into 50 μl of ice-cold competent cells in cuvettes (0.1cm) using a Bio-Rad gene pulser set at 1.75 kV, 25 μF with pulsecontroller set at 200 ohms. 1 ml of LB medium was added afterelectroporation. Cells were incubated at 32° C. for 1.5 hours withshaking and spread on appropriate selective or nonselective agar media.

[0307] Production of transgenic mice. Modified BAC and the p25-kbpsubclone DNAs were purified using cesium chloride gradients as described(Antoch et al., 1997). The 25-kbp subclone DNA was linearized by NotIdigestion before microinjection. BAC DNA (1 g/ml) and 25-kbp subcloneDNA (2 μg/ml) were microinjected into the pronucleus of (C3H/HeN-Mtv⁻ XC57BL/6Ncr) F₂ zygotes. Transgenic founders were subsequently identifiedby Southern analysis using a Cre probe or by PCR using primers5′CTGCTGGAAGATGGCGATTCTCG (SEQ ID NO: 36) and 5′AACAGCAGGAGCGGTGAGTC(SEQ ID NO: 37) that flank the 3′ insertional junction.

[0308] Histochemical analysis of β-galactosidase expression. Mice at 4to 5 weeks of age were sacrificed in CO₂ and perfused with 4%paraformaldehyde in PBS (pH 7.3). The brains, spinal cords and eyes wereremoved and postfixed for 3 hours. Vibratome sections (20 μm) of brainswere mounted on slides and used directly for X-gal staining or forimmunocytochemistry. For spinal cords and eyes, cryostat sections (20μm) were used that were made by cryoprotecting tissues in 30% sucrose inPBS overnight and embedding the tissues in freezing compound (OCT,Sakura). Before X-gal staining, samples on slides were postfixed with0.25% glutaraldehyde in PBS and briefly washed with rinse solution (0.1phosphate buffer pH7.3, 0.1% deoxycholic acid, 0.2% NP40 and 2mM MgCl₂).X-gal staining was performed by incubating samples in staining buffer(2.5 mg/ml X-gal, 5 mM potassium ferricyanide and 5 mM potassiumferrocyanide in staining buffer) for 2 hours at 37° C. followed bycounterstaining with 0.25% eosin (Fisher).

[0309] Immunocytochemistry. Immunostaining was carried out using the ABCVectastain kit (Vector Labs) on 20 μm vibratome sections. Sections wereblocked with PBS (pH7.3 containing 0.2% Triton X-100, 1.5% bovine serumalbumin and 5% normal goat serum) at room temperature for 2 hours andincubated with primary Eno2 antibody, a poly clonal rabbit anti-Eno2antiserum (Chemicon) at 1:100 dilution in PBS solution. After incubationwith a secondary biotinylated antibody and the ABC reagent, peroxidasewas reacted with 0.05% diaminobenzidine tetrahydrochloride (DAB) and0.003% hydrogen peroxide.

Example 18 Creation Of Improved Bacterial Host Strains forLambda-Mediated Recombination in BACs

[0310] Transfer of lambda recombination genes to DH10B cells.

[0311] To facilitate the use of lambda-mediated recombination with BACs,an improved phage-mediated recombination system has been created forefficient recombination using BACs. The DH10B strain unlike most otherstrains of E. coli is efficiently transformed with BAC DNA and containsmany of the BAC genomic libraries; it was judged to be a good hoststrain for subsequent modification.

[0312] Because DH10B is recA defective, standard genetic crosses cannotbe used to place the defective lambda prophage used for lambda-mediatedrecombination into the DH10B strain of E. coli. To circumvent thisproblem, DH10B was first converted to recA+, then the lambdarecombination genes were crossed in and the strain was again made recA−but now carrying the lambda genes.

[0313] To make DH10B recA+, a lambda transducing phage carrying therecA+gene was used to lysogenize DH10B creating the derivative DY303. Instrain DY330 used for λ mediated recombination, the tet gene conferringtetracycline resistance was inserted by homologous recombination wherethe cro-bioA deletion exists creating strain DY363. A P1 lysate made onDY363 was used to infect DY303 and by standard bacterial genetics thetet gene in the cro-bioA deletion was crossed into DY303. This deletes alarge segment of the λ DNA of the lysogen including the recA+ gene. Thisnew derivative of DH10B and DY303 is named DY380, is RecA− and carriesthe tet selectable marker substituted for the cro-bioA segment.. It wasobserved that DY380 cells were transformed with BAC DNA at efficienciesof 10⁻⁶ to 10⁻⁴.

[0314] Creation of DY380 Derivatives Containing Arabinose-inducible Creor flpe Genes

[0315] BAC targeting often makes use of a selectable marker to introducethe targeting cassette into the targeted locus. The selectable markercan, however, interfere with the subsequent function of the targetedlocus. To circumvent this problem, the inventors noted that a selectablemarker flanked with either frt or LoxP sites can be removed by eitherFlp or Cre recombinases, respectively. Thus, the inventors have createdtwo new strains, EL250 and EL350, by ultimately replacing the tet genein DY380 with araC and placing the flpe and Cre genes under anarabinose-inducible promoter. The genotypes of DY380, EL250, and EL350are shown schematically in FIG. 6. Although the arabinose-induciblepromoter p_(BAD) was used in this example, essentially any induciblepromoter may be used to activate flpe and Cre expression.

[0316] In DY380's prophage, tet is located between cI857 and bioA. InEL250's prophage,flpe replaces tet (flpe is a genetically engineered flpthat has a higher recombination efficiency than the original flp gene;Buchholz et al., Nature Biotechnology 16:657-662, 1998). Thus, asillustrated in FIG. 6, both EL250 and EL350 have heat-induciblehomologous recombination (the λ red genes) and arabinose-induciblesite-specific recombination (flpe or Cre) functions. This dualregulation allows both selective targeting by recombination as well asthe. subsequent removal of the selection marker from the targeted locusby site-specific recombination.

[0317] Improved Approach for Introducing Defective Lambda Prophages intoBacteria: Mini Lambda Circles

[0318] A method has been developed for introducing the λ-mediatedrecombination system directly into nearly any E. coli strain includingrecA defective DH10B derivatives. These derivatives can carry BACs,PACs, or other vectors.

[0319] The DY330 strain carries deletion of prophage genes from crothrough bioA. This deleted segment of λ and bioA were replaced to createa derivative that contains a fully normal λcI857 single-copy lysogen.Lysogens of this type can be induced at 42° C. to express λ functionsincluding the Red recombination functions. Because the λ carries all ofthe replication and lytic genes, induction for longer than 6 minutescauses death of cells carrying the lysogen. However, by inducing forless than 6 minutes, for example 4 minutes, recombination functions areonly partially activated, but cells survive when returned to grow at 32°C. Using, for example, a 4-minute time of induction, λ-mediatedrecombinants can be generated between linear, electroporated DNA and thechromosome including the DNA of the prophage. Thus, phage lambda itselfcan be used to lysogenize and generate recombinants in BAC strains.However, recombination efficiency would be low because of the shortinduction time.

[0320] PCR cassettes containing 5 genes for different drug resistancemarkers were amplified [cat, kan, amp, tet, spec (strep)] with flankinghomologies so as to replace prophage genes from cro through ea59 withthe respective drug markers selecting with that drug for resistantrecombinants at 32° C. A contiguous prophage DNA segment from baseposition 38,044 of the λ map in cro to base position 25,737 of the λ mapin sib are replaced by the drug cassettes (see Court and Oppenheim).This deletion eliminates all replication and lysis genes of the prophagecreating a defective prophage similar to that of the original DY330. Thedifference is that this prophage has both attachment sites attL and attRat the termini of the prophage whereas DY330 has attR through bioAdeleted as part of the cro-bioA deletion.

[0321] This set of strains (with respective drug cassettes) can beinduced for longer times than the complete lambda without killing thecells thereby providing maximal homologous recombination activity justas with DY330. The P_(L) operon of these prophages include the int andxis genes. Induction activates their expression and because both attLand attR are present causes site specific excision of the prophage as aDNA circle carrying its associated drug marker. Cells undergoinginduction for 15 minutes may lose the original prophage. This happens inabout 50% of the cells. The other 50% still have the prophage. The 50%with the prophage are likely to occur by reintegration of the circularDNA at the vacated attB site through Intmediated site specificrecombination.

[0322] The defective prophage DNA can be isolated and purified fromthese lysogens, if after a 15 minute induction, cells are lysed and DNAis isolated by plasmid purification protocols, i.e. by Qiagen columns.The circular phage DNA with its drug markers can be purified. This DNAcannot replicate upon retransfection into E. coli strains but it canexpress its pL operon and Int function to allow integration of thecircular DNA by site specific recombination between attP in the circularDNA and attB in the bacterial chromosome. Only Int and the host IHFfunctions are required for site-specific recombination. Such integratedDNAs are stable, are immune and can be selected by the drug marker eachcarries.

[0323] Because RecA is not required for site specific Int-mediatedrecombination, DH10B derivatives can be used for transformation and forintegration of the circular defective phage DNA selecting for itsappropriate drug marker.

[0324] The defective mini-prophage can also be induced as part of adi-lysogen in which a complete λ cI857 phage is also present. The phagelysate created by this 90 minutes induction at 42 degrees in L-Brothgenerates normal lambda phage particles as well as particles thatcontain the defective mini-prophage DNA (in λ terminology docLparticles). Infection of these lysates into cells (e.g. DH10B) allowsDNA injection of the mini-prophage DNA, site specific recombination, andselection for the drug marker carried on that DNA.

Example 19 An Improved Strategy and Improved Reagents for BACEngineering

[0325] To test the prophage system of Example 17 in BAC engineering, theefficiency of BAC recombination in EL250 cells was investigated. In theexperiments described in this example, a selectable cassette wastargeted to a mouse neuron-specific locus in a 250 kb BAC. The BAC wasthen further modified to enhance its usefulness in subsequent mousegenetic studies. These experiments validated an improved strategy andprovided improved reagents for BAC engineering using the lambdarecombination system.

[0326] The Eno2 gene is located in the middle of 284H12, a fullysequenced BAC (obtained from Research Genetics; Ansari-Lari et al.,Genome Research 8:29-40, 1998). The Eno2 gene was targeted because it isneural-specific and expressed in most mature neurons (Marangos andSchmechel, Annual Review of Neuroscience 10:269-295, 1987). By knockingout eno2 and replacing it with a Cre-containing cassette, a BACtransgenic line that expresses Cre in all mature neurons was created(described in Example 21). This BAC transgenic line is useful forsubsequent conditional knockout studies. The inventors used a BACapproach in part because BACs are large enough to contain all theimportant regulatory sequences required for proper regulation of geneexpression.

[0327] The following describes the construction of the BAC transgenicline with neuronal-specific Cre expression.

[0328] Generation of the Targeting Cassette and BAC-containing EL250Cells

[0329] The IRES-eGFPcre-FRT-kan-FRT targeting cassette was PCR amplifiedfrom pICGN21, which was constructed by subcloning a 1.9 kbpHindIII/AccI-digested and filled-in FRT-kan-FRT fragment from pFRTneointo the NotI/BcII-digested and filled-in cloning site of pIRESeGC. TheIRES-eGFPcre-FRT-kan-FRT cassette was amplified using chimeric 63 ntprimers. The 3′ 21 nt of each primer was homologous to the targetingcassette used for amplification while the 5′ 42 nt was homologous to thelast exon of Eno2 where the cassette was to be targeted byrecombination. The primers were designed to precisely target thecassette downstream of the Eno2 stop codon and upstream of its polyAsite.

[0330] The Eno2-containing 284H12 BAC was electroporated into EL250cells and six chloramphenicol resistant (Cm^(R)) colonies selected.Digestion of BAC DNA from six Cm^(R) colonies with EcoRI or HindIIIshowed that one had an abnormal digestion pattern. However, in other BACelectroporation experiments involving the analysis of more than 76additional colonies, no abnormal BACs were identified. These resultsindicate that BAC rearrangements during electroporation are rare.Subsequent experiments were carried out with Cm^(R)-resistant EL250colonies harboring BACs having proper EcoRI and/or HindIII digestionpatterns.

[0331] Generating and Isolating a BAC with a Disrupted eno2 Locus

[0332] Next, the 284H12 BAC was modified to disrupt the eno2 locus withthe IRES-eGFPcre-FRT-kan-FRT targeting cassette. The methods used inthese experiments were similar to those described extensively herein(and in Yu et al., Proc. Natl. Acad. Sci. U.S.A. 97:5978-5983, 2000).The approach is illustrated schematically in FIG. 7.

[0333] EL250 cells carrying the 284H12 BAC were shifted to 42° C. for 15minutes to induce lambda Exo, Beta, and Gam expression. The cells werethen electroporated with 300 ng of the amplifiedIRES-eGFPcre-FRT-kan-FRT cassette, and kanamycin-resistant (KMR)colonies were selected. A kanamycin-resistant phenotype indicated thatthe targeting cassette was successfully integrated into the 284H12 BAC(illustrated as “Targeting,” FIG. 7, middle). Approximately 5200 Km^(R)colonies were obtained from 10⁸ electroporated cells for a targetingefficiency of about 10⁻⁵. No colonies were obtained from control cellsthat were not heat-induced. Thus, lambda recombinase expression wasrequired for efficient recombination.

[0334] Twenty-four kanamycin resistant colonies were analyzed withwhole-cell PCR using primers that flanked the targeted locus. The PCRresults indicated that all were correctly targeted. Sequencing of thetargeted region from six colonies, however, showed that three carriedpoint mutations. To determine whether these point mutations wereintroduced during PCR amplification or during homologous recombination,the targeting was repeated. This time, however, the PCR-amplifiedIRES-eGFPcre-FRT-kan-FRT cassette was subcloned into the SmaI site ofpBluescript by blunt-end ligation before targeting, and plasmidscarrying wild type amplified cassettes identified by DNA sequencing.These cassettes were then released from the plasmid by BamHI digestionand used for targeting. Using this two-step method, all twelve targetedBACs that were subsequently sequenced contained wild typeIRES-eGFPcre-FRT-kan-FRT cassettes. These results indicate that thepoint mutations were introduced by the primers used or during PCRamplification of the targeting cassette rather than during targeting.

[0335] Removing the Kanamycin-resistance Marker

[0336] Next, the kan selectable marker was removed from the BAC toprevent it from possibly interfering with Cre expression. This processwas initiated by arabinose treatment, which induces EL250 cells toexpress the Flpe recombinase. The process is illustrated in FIG. 7,bottom line (“Flip-out of kan”).

[0337] Overnight cultures from single Km^(R) colonies were diluted50-fold in LB medium and grown till OD₆₀₀=0.5. Flpe expression from theEL250 cells was then induced by incubating the cultures with 0.1%L-arabinose for 1 hour. The bacterial cells were subsequently diluted10-fold in LB medium, grown for an additional hour, and spread onchloramphenicol plates (12.5 ug/ml). The next day, 100 Cm^(R) colonieswere picked and replated on kan plates (25 ug/ml) to test for loss ofkanamycin resistance. Chloramphenicol resistance indicates that the cellretained the BAC, whereas kanamycin sensitivity indicates that kan hasbeen successfully removed from the BAC. All colonies were Km^(s) andcontained a single frt site at the targeted locus.

[0338] Without being bound by theory, it is likely that the surprisinglyhigh recombination efficiency reflects the tight control of Flpeexpression afforded by the single copy P_(BAD) promoter and flpe gene,and the fact that the frt sites are located in cis rather than in transto each other.

[0339] Removing an Undesirable LoxP Site in the BAC Vector Backbone

[0340] A LoxP site contained in the BAC vector backbone (pBeloBAC11;Shizuya et al., Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797, 1992) wasremoved by a final round of gene targeting.

[0341] To facilitate the removal of this undesirable LoxP site, a newplasmid, pTamp, was constructed that contains an amp gene flanked by 920bp of pBeloBAC11 sequence located 5′ of the LoxP site and 370 bp ofpBeloBAC11 sequence located 3′ of the LoxP site. This amp insert can bereleased from pTamp by BamHI digestion and used to replace the LoxP sitein the BAC transgene by gene targeting. This targeting reaction is veryefficient due to the large amount of homology between the amp cassetteand the pBeloBAC11 vector (56,200 colonies per 10⁸ electroporatedcells).

[0342] Upon removal of the undesirable LoxP site, the modified 284H12BAC was used in the transgenic mouse studies described in Example 21.

Example 20 Subcloning by GAP Repair

[0343] This λ-mediated recombination system can also be used to subclonefragments from BACs without the use of restriction enzymes or DNAligases. This form of subcloning relies on gap repair to recombine thefree ends of a linear plasmid vector with homologous sequences carriedon the BAC (FIG. 8). The method is readily adaptable to other forms ofintramolecular and extrachromosomal DNA, such as plasmids, yeastartificial chromosomes, P1 artificial chromosomes, and cosmids. Thisnovel method combines lambda mediated recombination with gap repair toenable recombination of very large DNA segments onto an extrachromosomalvector.

[0344] The linear plasmid vector with an amp selectable marker and anorigin of replication carries the recombinogenic ends (FIG. 8B). Thevector is generated by PCR amplification using two chimeric primers. The5′ 45-52 nt of each primer is homologous to the two ends of the BACsequence to be subcloned while the 3′ 20 nt is homologous to plasmidDNA. Recombination generates a circular plasmid in which the DNA insertwas retrieved from the BAC DNA via gap repair. Circular plasmids areselected by their Amp^(R).

[0345] To determine the maximum sized fragment that can be subclonedfrom BACs using this method, several different pairs of primers weregenerated in which the homology segments were located 25 kb, 48 kb, 60kb, or 80 kb apart in the Eno2 BAC DNA (FIG. 8A). Rare cutter NotI andSalI restriction sites were also incorporated into these primers so thatthe subcloned fragments could be released from the recombinant clonesintact. Using pBluescript as the cloning vector, it was possible tosubclone the 25 kb fragment. However, attempts to subclone largerfragments were unsuccessful. As a possible explanation for this result,it was hypothesized that sulfones containing larger fragments on a highcopy vector such as pBluescript were toxic to the cell.

[0346] To determine if the hypothesis was correct, a lower copy numbervector (pBR322, with its copy number control element intact) was used asthe cloning vector. Fragments as large as 80 kb could be subcloned witha pBR322 vector. Not all subclones obtained by gap repair had thecorrect inserts (as determined by restriction enzyme pattern analysis).Some subclones lacked inserts while others contained inserts withaberrant restriction patterns. In order to confirm that the correctinsert has been subcloned, when using subcloning by gap repair, a methodof screening subclones can be used to assure that the selected subclonedcontains the desired insert. Such methods include restriction mapping,sequencing, PCR analysis, Southern analysis, etc., and other methodswell known to those of skill in the art.

[0347] The ability to subclone large fragments of genomic DNA by gaprepair should facilitate many studies in genome research that weredifficult or impossible to perform previously. For example, Gap repairfor cloning on to vectors can be used with many different vectors usedfor protein expression in bacteria, plants and animal cells,mutagenesis, cloning, transcription, etc. Targeting vectors ortransgenic constructs can be subcloned with ease, and virtually anyregion of the engineered BAC can now be included in the desiredsubclone.

[0348] Lambda mediated recombination combined with gap repair makes itpossible to subclone fragments from complex mixtures without firstpurifying the DNA to be subcloned. This greatly facilitates thesubcloning process and allows for high throughput subcloning of tens ofthousands of genes or DNA molecules into many different vectorbackbones. This will greatly facilitate studies designed to determinethe function of genes uncovered in large scale sequencing projects. Forexample, cDNA clones for genes of unknown function can be subcloned intomany different expression vectors and the function of these genesstudies in cell-based assays in vitro or in the whole animal. This typeof subcloning does not rely on PCR amplification, which can introduceunwanted mutations into the subcloned sequences.

[0349] Subcloning by gap repair also facilitates the identification oflocus control regions or other regulatory elements that may be locatedat some distance from the gene. Many such potential elements arepresently being identified by techniques such as comparative genomesequencing. Examples include pathogenicity islands, replicative originsand segregation elements. The ability to modify precisely theseregulatory sequences on BACs, combined with the ability to include orexclude them during the subcloning process, will make it possible todissect the function of these sequences in the whole animal or in vitroat a level not previously possible.

Example 21 Production of Transgenic Mice Using BACs

[0350] Examples 18-20 describe the construction of a modified BACbelieved to contain all of the regulatory sequences needed forneural-specific Cre expression in transgenic mice. To investigate thishypothesis, the modified BAC described in Example 18 was injected into(C3H/HeN-Mtv⁻ X C57BL/6Ncr) F₂ zygotes. A BAC transgenic line carryingapproximately two copies of the transgene was then established.

[0351] In addition to the BAC transgenic line, two transgenic linescarrying 25-kbp subclones of the BAC were also established. The 25-kbpsubclones contains the entire modified Eno2 coding region as well as 10kbp of 5′ and 5 kbp of 3′ flanking sequences, respectively. Onetransgenic line, 25 kbp-1 carries approximately four copies of thetransgene, while the second, 25 kbp-2 carries approximately five copiesof the transgene. Thus, Cre expression in the BAC transgenic line couldbe compared to Cre expression in the transgenic lines carrying thesubclone.

[0352] The transgenic mice were crossed to ROSA26 reporter mice, whichcontain a lacZ reporter that can be activated by Cre recombinase(Soriano, Nature Genetics 221:70-71, 1999). Double heterozygotes weresubsequently analyzed by X-gal staining at 4 weeks of age.

[0353] Several different tissues were examined for X-gal expressionincluding the brain, spinal cord, eye, lung, heart, intestine, muscle,liver, spleen, and kidney. Blue stained cells were found only in neuraltissue in the three transgenic lines, indicating that both the BAC andthe 25-kbp subclone contain the regulatory elements needed forneural-specific expression. The pattern of Cre activity was, however,different in the three lines. Vibratome sections of the brain from theBAC transgenic mice showed blue-stained cells throughout the gray matterbut not in the white matter, indicative of Cre activity in most neuronsbut not in glial cells. In contrast, X-gal staining in the 25 kbp-1 and25 kbp-2 transgenic mice was present in only a subset of neurons andexpression was variable between the two different lines.

[0354] Higher power magnification of the cerebellum of the BACtransgenic mice showed that Cre was expressed in virtually all neuronalcells. This included Purkinje cells in the Purkinje cell layer, granuleand Golgi cells in the granular layer, basket cells and stellate cellsin the molecular layer and neurons of the deep cerebellar nuclei. Incontrast, in the 25 kbp-1 line, Cre was expressed in only a subset ofGolgi cells in addition to a few cells in the granule and Purkinje celllayers. Glial cells of white matter also expressed Cre indicative ofleaky expression. In the 25 kbp-2 line, Cre expression was limited tothe gray matter and included a variety of neuronal cell types, includingmost basket cells, stellate cells, Purkinje cells and neurons of thedeep cerebellar nuclei. In contrast, few granule cells and Golgi cellsin the granule layer expressed Cre.

[0355] Higher power magnification of the hippocampus and cortex showedsimilar results. In the hippocampus of BAC transgenic mice, virtuallyall neurons in the comu Ammonis (CA) region and the dentate gyrus (DG)expressed Cre. The same was true in the cortex, where all six layers ofthe cortex that contained neurons (layers II-VI) expressed Cre. Incontrast, the hippocampus of 25 kbp-1 transgenic mice showed reduced Creexpression in the DG (FIG. 4E) and layers II and III of cortex. The 25kbp-2 transgenic mice showed even lower levels of Cre expression in theDG. The CA1 and CA2 regions of the CA also failed to express Cre. Creexpression was also greatly reduced in the cortex, with layers II andIII showing most the reduction.

[0356] Cre activity in the spinal cord, dorsal root ganglion (DRG) andretina of the transgenic mice was also examined in order to determinewhether Cre was expressed in mature neurons within the peripheralnervous system. Similar to what was observed for the central nervoussystem, Cre was expressed in most mature peripheral neurons in the BACtransgenic mice while fewer peripheral neurons expressed Cre in the two25 kbp transgenic lines.

[0357] To determine whether Cre was expressed in all Eno2protein-positive neurons, a section from the brain of a BAC transgenicanimal was immunostained with an anti-Eno2 antibody followed by X-galstaining for Cre activity. Virtually all Eno2-positive neurons wereactive for Cre. Thus, Cre expression in BAC transgenic animalscorrelated tightly with native mouse Eno2 promoter-enhancer activity.

[0358] The present application, particularly Examples 17-20, describes ahighly efficient recombination system for manipulating BAC DNA in E.coli. The recombination system uses a defective λ prophage to supplyfunctions that protect and recombine the electroporated linear DNAtargeting cassette with the BAC sequence. Because the recombinationfunctions are expressed from a defective prophage rather that a plasmid,the recombination functions are not lost during cell growth as oftenhappens with plasmid-based systems. Another advantage of this prophagesystem is that the λ gam and red recombination genes are under thecontrol of the temperature sensitive λ repressor that provides a muchtighter control of gam and red expression than can be obtained onplasmids. This tight regulation, combined with the strong λ pL promoter,which drives gam and red expression to very high levels, makes itpossible to achieve recombination frequencies that are surprisinglyefficient (at least 50-100 fold higher than those obtained withplasmid-based systems; Narayanan et al., Gene Therapy 6:446:442-447,1999; Muyers et al., Nucleic Acids Research 27:1555-1557, 1999).The tight control prevents expression of any recombination functionsexcept for the 15 minute temperature induction.

[0359] The ability to precisely manipulate large fragments of genomicDNA, independent of the location of appropriate restriction enzymesites, has many applications for functional genomics, both in the mouseand in other organisms. As shown herein, Cre can be introduced into thecoding regions of genes carried on BACs facilitating the generation ofCre-expressing transgenic lines for use in conditional knockout studiesor for use in conditional gene expression studies. Genes can also beepitope tagged and microinjected into the germline of mice carrying amutation in the gene. If the epitope tagged transgene rescues the mutantphenotype, the epitope tagged protein is functional and the epitope tagcan serve as a marker for expression of the gene. Likewise, a genecarried on a BAC can be replaced with another gene and the function ofthe “knock-in” mutation assayed in transgenic mice.

[0360] This recombination system also facilitates the generation ofcomplicated conditional targeting vectors. While the generation of suchvectors often used to take several months it can now be done in a onlyfew weeks time. The ability to reversibly express Cre or Flperecombinases in E. coli speeds this process even further. Moreover, asdemonstrated in Example 18, a selectable marker flanked with LoxP or frtsites can be now be introduced into an intron of a gene and then removedby transient Cre or Flpe expression leaving behind a solo LoxP or frtsite in the intron (see also Examples 24-27).

Example 22 BAC Recombination without Drug Selection

[0361] The high efficiency of recombination described in Example 18 andelsewhere in these examples suggested that targeting could be donewithout drug selection. Direct targeting without drug selection wouldoffer a number of significant advantages. In particular, it wouldfacilitate genomic experiments in which the presence of a selectablemarker, or even a frt or LoxP, scar might be undesirable.

[0362] To demonstrate that targeting can be achieved without drugselection, a 24 bp FLAG tag was targeted to the 5′ end of the SRY-boxcontaining gene 4 (Sox4 ) gene carried on a 125 kb BAC. For theseexperiments, a 114 bp targeting cassette was generated in which two45-bp arms homologous to the Sox4 gene flanked the 24-bp FLAG sequence.This DNA fragment was created by synthesizing two 79-bp oligonucleotidesthat overlapped at their 3′ ends by 44 bp. These overlaps were annealedand filled in by Taq polymerase.

[0363] Expression of lambda recombinase genes from the defectiveprophage was heat-induced in DY380 cells carrying the Sox4 BAC. Then,the FLAG-tagged cassette was introduced into the cells byelectroporation. The cells were then spread on LB plates to a density of˜2,000 cells per plate. Colonies containing the FLAG tag weresubsequently identified by colony hybridization using a 30-bpFLAG-specific oligonucleotide probe (24 bp FLAG tag and 3 bp on eachside that was homologous to the Sox4 targeted site).

[0364] Among 3,800 colonies screened from uninduced cells, noFLAG-positive colonies were identified. In contrast, seven FLAG-positivecolonies were identified in 4,210 colonies obtained from induced cellsfor an overall targeting frequency of 1.7×10⁻³. PCR amplification anddirect sequencing showed that each of the seven FLAG-positive colonieswas correctly targeted.

[0365] As unequivocally demonstrated in this example, the surprisinglyhigh recombination efficiency offered by this recombination system makesit possible to manipulate BAC or other DNA without drug selection. Pointmutations, deletions, or insertions can now be engineered into any geneon a BAC in the absence of a confounding linked drug selection marker ora LoxP or frt site. In cases where the gene is mutated in human disease,the exact disease-causing mutations can be engineered on the BAC and theeffect of these mutations analyzed in transgenic mice.

Example 23 Materials and Methods for Examples 24-27

[0366] Bacterial Strains: The E. coli strains used in Examples 24-27 arelisted in Table 9, below. TABLE 9 Recombineering reagents StrainsGenotype DH10B F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) Ø80dlacZΔM15 ΔlacX74 deoRrecA1 endA1 araD139 Δ(ara, leu)7649 galU galK rspL nupG DY380 DH10B [λcI857 (cro-bioA <> tet] EL250 DH10B [[λ cI857 (cro-bioA <>araC-P_(BAD)flpe] EL350 DH10B [[λ cI857 (cro-bioA <> araC-P_(BAD)cre]Selection Cassettes PL451 FRT-PGK-EM7-NeobpA-FRT-loxP PL452LoxP-PGK-EM7-NeobpA-loxP Other Plasmids pSK+ pBluescript PL253 ModifiedMC1TK

[0367] EL350 cells were derived by transferring the defective λ prophagepresent in DY330 cells (Yu et al., Proc Natl Acad Sci U.S.A.97:5978-5983, 2000) into DH10B cells, to create DY380 cells (Lee et al.,Genomics 73: 56-65, 2001). An arabinose-inducible Cre gene (P_(BAD)-cre)was then introduced into the defective λ prophage present in DY380 cellsto create EL350 cells (Lee et al., supra, 2001). DH10B cells have beenused to construct most BAC libraries and are highly permissive for BACtransformation, while DY330 cells are relatively resistant to BACtransformation. BACs were identified from the CITB BAC libraryconstructed from CJ7 (129/Sv) ES cells (Research Genetics). DH10Belectrocompetent cells were purchased from Invitrogen.

[0368] Construction of Retrieval and Targeting Vectors: PCR primers weredesigned using Mac Vector. Primer sequences used for constructing theEvi9 conditional knockout vector are listed below: Primer A:NotIEvi9-ex4-Ret-5′-1, 5′-ATAAGCGGCCGCTCTAATACAGAC-TGGCACCTG-3′; (SEQ IDNO: 38) Primer B: H3Evi9-ex4-ret-5′-2,5′-GTCAAGCTTTAAAGA-GATCCCTGCTATAAA-3′; (SEQ ID NO: 39) Primer Y:H3Evi9-ex4-Ret-3′-1, 5′-GTCAAGCTTCCTGTTTCCAGCGTAG-GTGAA-3′; (SEQ ID NO:40) Primer Z: SpeIEvi9-ex4-ret-3′-2,5′-TCTACTAGTCTCACC-ACCTGTACAGTAAGT-3′; (SEQ ID NO: 41) Primer C:NotIEvi9-ex4-5′L-1, 5′ATAAGCGGCC-GCAACAATTAGTGTGTTTCCAGTT-3′; (SEQ IDNO: 42) Primer D: EcoRI-BgIII-Evi9-ex4-5′L-2,5′-GTCGAATTCAGATCTAAATGG-GGTACTGAGACAAG-3′; (SEQ ID NO:44) Primer E:BamHIEvi9-ex4-5′R-1, 5′-ATAGGATC-CAACCAATGAGACAGTGGCACA-3′; (SEQ ID NO:45) Primer F: SalIEvi9-ex4-5′R-2,5′-GTC-GTCGCACTTATTCATGTTCCAAC-AA-CCA-3; (SEQ ID NO: 46) Primer G:NotIEvi9-ex4-3′L-1 5′-ATAAGCGGCCGCCTTAACT-TAGACAGCATGTAT-3′, (SEQ ID NO:47) Primer H: EcoRI-Evi9-exon4-3′L-2,5′-GTCGAAT-TCGTCTGCAGAGGGTTAGTCAA-3′; (SEQ ID NO: 48) Primer I:BamHI-Evi9-ex4-3′R-1, 5′-ATAGGATCCAGAGCAGATAGCAGTGAAAA-3′; (SEQ ID NO:49) Primer J: SalIEvi9-ex4-3′R-2, 5′GTCGTCGCATATTAGCTCACCCAATGC-TA-G-3′.(SEQ ID NO: 50)

[0369] These primers amplify the following size fragments: 500 bp withprimers A, B; 295 bp with primers Y, Z; 222 bp with primers C, D; 276 bpwith primers E, F; 277 bp with primers G, H; and 227 bp with primers I,J.

[0370] PCR amplification: (ROCHE Expand High-Fidelity Taq kit) wasperformed by setting up the first reaction mixture containing 1 μl dNTP(10 mM), 1 μl DNA (10 ng BAC DNA), 1 μl (10 μM) of each primer, and 21μl water. Then, a second reaction mixture was set up that contained 5 μlof 10× PCR buffer (#2), 0.75 μl high-fidelity Taq (5 u/μl), and 20 μlwater. The two reaction mixtures were then combined. PCR was performedusing a PE-9700 PCR machine with the following settings: 94° C. for 2minutes, then 10 cycles of 94° C. for 15 seconds, 55° C. for 30 seconds,70° C. for 1 minutes. This was followed by 15 cycles of 94° C. for 15seconds, 55° C. for 30 seconds, 70° C. for 1 minutes, with an additional5 sec extension time each cycle. 5 μl of the 50 μl PCR reaction mixturewas loaded onto a gel to check the PCR reaction. The remaining 45 μl wasmixed with 225 μl PB from Qiagen and loaded onto a Qiagenmini-preparation spin column. After a 30-second spin, the column waswashed once with 750 μl PE buffer. The PCR fragments were eluted using30 μl of EB from Qiagen. 3 μl of restriction buffer (10×) and 1 μl ofrestriction enzyme was added and the mixture incubated at 37° C. for 1hour. The digested PCR fragments were purified again with the columnsand were ready for ligation.

[0371] The retrieval vector was generated by mixing 3 μl of PCR product1 (left arm, NotI/HindIII), 3 μl PCR product 2 (right arm,HindIII/SpeI), 2 μl MC1TK (PL253) (NotI/SpeI, 1 μl 10× ligation bufferand 1 μl T4 DNA ligase.

[0372] The Neo-targeting vector was generated by mixing 3 μl of PCRproduct 1 (left arm, NotI/EcoRI), 3 μl PCR product 2 (right arm,BamHI/SalI), 2 μl floxed Neo cassette (PL452 or PL451) (EcoRI/BamHI), 1μl pSK+(NotI/SalI), 1.2 μl 10× ligation buffer and 1 μl T4 DNA ligase.The ligation mixtures were incubated at 16° C. for 2 hours and 0.5 μlwas transformed into electro-competent DH10B cells (Invitrogen).

[0373] Transformation of BAC or Plasmid DNA into Recombinogenic Strains:E. coli cells with BACs were grown overnight in 5 ml LB broth withchloramphenicol. The LB broth used in contained only 5 g NaCI per liter.Cells were collected in three eppendorf tubes (2 ml) and wereresuspended in 250 μl P1 from Qiagen. 250 μl P2 and 350 μl P3 were thenadded to each tube and the tubes spun for 4 minutes. The supernatantfluid from these tubes was transferred to new 1.5 ml eppendorf tubes,which were spun for another 4 minutes to clear the supernatant fluids.Finally, 750 μl isopropanol was added to precipitate the DNA (roomtemperature for 10 minutes) and the DNA collected by spinning the tubesfor 10 minutes at the maximal speed. The DNA pellet was washed once with1.0 ml 70% ethanol, dried and resuspended in 50 μl TE (total from 3tubes). 1 μI DNA was used for electroporation and 10 μl for digestion(20 ng RNase was added to clear the RNA). Only freshly prepared BAC DNAwas used for transformation.

[0374] EL350 or DY380 cells were grown in 5 ml LB broth in a Falcon 14ml polypropylene round-bottom tube at 32° C. overnight with shaking. Thenext day the cells (OD600=1.2) were collected by centrifuging at 4000rpm (0° C.) for 5 minutes in Oak Ridge tubes. Cell pellets wereresuspended in 888 μl ice-cold water. Cells were transferred to a 1.5 mleppendorf tube (on ice) and centrifuged using a benchtop centrifuge for15-20 seconds at room temperature. The tubes were placed on ice and thesupernatant fluids aspirated. The process was repeated two more times.Finally, the cell pellet was resuspended in 50 μl ice-cold water andtransferred to a pre-cooled electroporation cuvette (0.1 cm gap). 1 μlBAC DNA (100 ng) or plasmid DNA (1.0 ng) was added and mixed.Electroporation was performed using a BIO-RAD electroporator under thefollowing condition: 1.75 kV, 25 uF with pulse controller set at 200ohms. The time constant was usually set at 4.0. 1.0 ml LB was added toeach cuvette, which was incubated at 32° C. for one hour. Cells werespread on plates with the appropriate antibiotics.

[0375] Retrieving: EL350 cells containing BAC-A12 were inoculated into 5ml of LB broth in a Falcon 14 ml polypropylene round-bottom tube andgrown at 32° C. overnight with shaking. The next day, 1.0 ml of theovernight culture (OD600=1.2) was transferred to 20 ml LB(OD600=0.05-0.1) and incubated for 2 hours with shaking (180 rpm,OD600=0.5). 10 ml of the cells were then transferred to a new flask andshaken in a 42° C. water bath for 15 minutes. The cells were put intowet ice and the flask shaken to make sure that the temperature of theflask dropped as fast as possible. The flask was left in wet ice foranother 5 minutes. The cells were transferred to 25 ml glass centrifugetubes and spun at 4000 rpm (0° C.) for 5 minutes (with rubber adaptors).Cells were resuspended in 888 μl ice-cold water and transferred to a 1.5ml eppendorf tube (on ice) and washed three times with ice-cold water asdescribed above. Finally, the cell pellet was resuspended in 50 μlice-cold water. 1-2 μl of the purified PCR or plasmid fragment was addedand electroporated as described above.

[0376] Targeting: Frozen EL350 electro-competent cells were used fortargeting in co-electroporation. The frozen cells were produced byadding a 10 ml overnight culture of EL350 (grown in two 14 ml tubes,OD600=1.2) to 500 ml LB broth in a 2-liter flask. The culture was thenplaced in a waterbath shaker at 32° C. until OD600=0.5 (˜2.0 hour). Theflask was then transferred to a 42° C. waterbath shaker and incubatedfor 15 minutes. The flask was immediately put into an ice slurry andshaken for 5 minutes by hand to make sure the temperature dropped asfast as possible. The flask was put on ice for an additional 10 minutes.Cells were collected at 4000 rpm at 0° C. for 5 minutes and washed threetimes with ice-cold water and once with cold 15% glycerol in water.Finally, cells were resuspended in 4 ml ice-cold 15% glycerol in water.50 μl of the cells were aliquoted to pre-cooled eppendorf tubes (80tubes total) and stored at −80° C.

[0377] For electroporation, the frozen cells were thawed at roomtemperature and quickly put on ice. Co-transformation of the purifiedtargeting cassette (100 ng in 1 μl EB) and the template plasmid DNA (10ng in 1 μl EB) was performed using with a BIO-RAD electroporator asdescribed previously.

[0378] Excision of the Neo Cassette: Frozen EL3 50 cells induced for Creexpression by prior growth in arabinose-containing medium were used forexcision of the floxed Neo cassette. A 10 ml overnight culture of EL350cells was added to 500 ml of LB broth in a 2-liter flask. The culturewas placed in a water bath shaker at 32° C. until OD600=0.4 (2.0 hours,180 rpm). 5 ml of 10% L(+)arabinose (Sigma A-3256) in H₂O was added tothe culture to a final concentration of 0.1% and shaken at 32° C. foranother hour. Cells were collected, cell pellets washed, and frozen asdescribed above. 1 ng plasmid DNA was electroporated into 50 μl frozencompetent cells. 1.0 ml LB broth was added to the electroporationcuvette. 10-100 μl of the cells were subsequently plated on anampicillin plate and 100 μl on a kanamycin plate and incubated at 32° C.overnight. The ampicillin plate ideally has 10-100 colonies, and nocolonies on the kanamycin plate. The following antibiotic concentrationswere used in the experiments: kanamycin and chloramphenicol, 12.5 μg/mlfor BACs, 25 μg/ml for multicopy plasmids; Ampicillin, 25 μg/ml forBACs, 100 μg/ml for pBluescript.

[0379] Gene Targeting in Mouse ES Cells: 20 μg NotI-linearized Evi9cko-targeting vector (PL460) DNA was electroporated into 10×10⁶ CJ7 EScells that were growing on mitomycin-C-inactivated STO cells.Transfectants were selected in M15 medium (15% fetal bovine serum inDMEM with 2 mM L-glutamine) with G418 (180 μg/ml) and ganciclovir (2μM). Targeted clones were identified on Southern blots with the 5′ and3′ probes.

Example 24 Subcloning DNA by GAP Repair

[0380] Conditional knockout (cko) targeting vectors can be made by usingrecombineering to introduce LoxP sites, and positive and negativeselection markers, into BAC DNA by homologous recombination. The regionof the BAC containing the LoxP sites, and positive and negativeselection markers, is then excised from the BAC and transformed into EScells. The introduction of LoxP sites into BACs is complicated, however,because most BAC vector backbones carry Lox sites. These sites must beremoved before any further Lox sites are introduced into the BAC DNA.Additionally, BAC integrity needs to be examined after eachmodification, and this is difficult when the BAC inserts are large. Bysubcloning a 10-15 kb fragment of BAC DNA into a high copy plasmidvector such as pBluescript (pSK+) before the Lox sites are introduced,these problems can be eliminated.

[0381] Homologous recombination via a process known as gap repairprovides a convenient method for subcloning DNA from BACs intopBluescript. The gap repair method used previously for subcloning BACDNA is shown in FIG. 14. Here, the linearized pBluescript vector usedfor gap repair is generated by PCR amplification using two chimericprimers (Zhang et al., Nat Genet 30: 31-39, 2000; Lee et al., Genomics73:56-65 2001). The 5′ 50 nucleotides of each primer are homologous tothe two ends of the BAC sequence to be subcloned, while the 3′ 20nucleotides of each primer are homologous to pBluescript DNA. Thelinearized, PCR-amplified pBluescript vector is electroporated into E.coli cells induced for exo, bet, and gam expression, and which carry theBAC. Homologous recombination between the BAC DNA and the linearizedpBluescript vector generates a circular plasmid that can replicate in E.coli. Ampicillin resistance (Amp^(r)) can be used to select thesecircular products (FIG. 14).

[0382] In order to make subcloning by GAP repair possible, a BAC must befirst transferred from its strain of origin (DH10B) into an E. colistrain that contains exo, bet, and gam. In the experiments describedherein, BACs are transferred into EL350 E. coli cells (Examples 20-21).EL350 cells were made by constructing a defective lambda prophage inDH10B cells, to create DY380 cells (Example 18) since DH10B is one ofthe few E. coli strains known that can be efficiently transformed withBAC DNA. A Cre gene under the control of the arabinose induciblepromoter, PBAD, was then introduced into the defective prophage carriedin DY380 cells, to produce EL350 cells (Lee et al., Genomics 73:56-65,2001). In EL350 cells, the homologous recombination functions encoded bythe red genes can be controlled by temperature, while the Cre gene canbe controlled by arabinose. As disclosed herein, it is much easier totransform electro-competent EL350 or DY380 cells produced from overnightcultures, than from exponentially growing cells. When BAC DNA iselectroporated into stationary electro-competent cells and theBAC-containing cells selected using the chloramphenicol resistance(Cam^(r)) gene that is carried in the BAC vector backbone, 100 to 1000Cam^(r) colonies are routinely obtained from 50 ng of BAC DNA, andvirtually all of the colonies contain unrearranged BACs. A complete listof the reagents used in these studies can be found in Table 9.

[0383] An alternative method was used to subclone an 11.0 kb fragment ofEvi9 spanning exon4, an alternative method for generating gap-repairedplasmids was designed that makes use of longer homology arms (200-500bp; FIG. 17). As shown below, these larger homology arms significantlyincrease the frequency of subcloning by gap repair, and because of this,unwanted recombination products were rare. Another advantage of thisalternative method is that the gap repair plasmid is not PCR amplified,which eliminates potential PCR artifacts introduced into the plasmid byPCR. In this alternative method, two sets of PCR primers were producedand used to amplify two 200-500 bp regions of the BAC (primers A and Band Y and Z; FIG. 15). Ultimately these two regions will mark the endsof the fragment to be subcloned by gap repair. The PCR products werepurified using spin columns and digested with either NotI and HindIII orHindIII and SpeI. Restriction sites for these enzymes were included inthe amplification primers in order to permit directional cloning of thePCR products into pBluescript. The digested-fragments were againpurified and ligated to NotI- and SpeI-cut pBluescript DNA that also hasa TK gene (MC1TK) gene for use in negative selection in ES cells. Theretrieval vector was subsequently linearized with HindIII to create aDNA double strand break for gap repair.

[0384] When 1 μl (50-100 ng) of the linear gap repair plasmid waselectroporated into electro-competent EL350 cells, which contained Evi9BAC A12, and which had been induced for exo, bet, and gam expression byprior growth at 42° C. for 15 minutes (FIG. 14), it was found thatseveral thousand Amp^(r) colonies were routinely generated in a singleelectroporation experiment. About 5% of these Amp^(r) colonies werebackground colonies derived either from self-ligation of the linearizedgap repair plasmid or from uncut DNA. The other 95% of the coloniescontained gap-repaired plasmids with the expected genomic inserts (FIG.16B, lane 1).

[0385] During the gap repair process, RecBCD is inhibited by Gam so thatthe linear gap repair plasmid is stable. However, in the absence ofRecBCD, Co1E1-derivative plasmids such as pBluescript can replicate byrolling circle replication. This type of replication will eventuallyconvert the plasmid monomers into plasmid multimers (Feiss et al. Gene17:123-130, 1982). As a result, huge plasmid complexes are produced inRecBCD-deficient cells. To select against these plasmid multimersfollowing gap repair, a small amount of the gap-repaired plasmid DNA (1ng) was re-transformed into wild type DH10B cells, and Amp^(r) coloniesselected. Empirically, it was determined that re-transformation selectsfor plasmids monomers and eliminates plasmid multimers.

Example 25 Targeting the First LoxP Site into the Subcloned Plasmid DNA

[0386] The next step in creating a cko-targeting vector is theintroduction of a LoxP site into the subcloned DNA: in this case, 5′ ofEvi9 exon 4 (FIG. 15A). This is accomplished by introducing a floxedneomycin resistance (Neo) cassette (PL452) via homologous recombinationinto the subcloned plasmid DNA, and by removing the Neo gene via Crerecombinase. The floxed Neo gene in PL452 is expressed from a hybridPGK-EM7 promoter. PGK permits efficient Neo expression in mammaliancells, while EM7 allows Neo to be expressed in bacterial cells.Subsequent removal of the floxed Neo gene via Cre recombinase leavesbehind a single LoxP site at the targeted locus. In order to introduce afloxed Neo gene at the correct location, it is first flanked with100-300 bp arms that are homologous to the targeting site. Thesehomology arms, as described above, are generated by PCR amplification ofthe BAC DNA. In this case, the PCR primer pairs were engineered tocontain NotI and EcoRI (primers C and D) or BamHI and SalI (primers Eand F) restriction sites (FIG. 14). These restriction sites allow forthe directional cloning of the homology arms, and the floxed Neo gene,into pBluescript. Primer D also contains a BglII site internal to theEcoRI site. The BglII site marks the presence of the LoxP site at thetargeted locus following recombination in ES cells (see below). An EcoRVsite was also incorporated into primer G for 3′ side diagnosis of thetargeting in ES cells (see below). Following PCR amplification, theproducts were purified, restriction digested and ligated to the floxedNeo cassette excised from PL452 with EcoRI and BamHI, and to pBluescriptthat was linearized by NotI and SalI digestion (FIG. 14). Four to sixcolonies selected by their kanamycin resistance, conferred by Neo, werepicked and checked by restriction enzyme digestion to ensure that theywere properly constructed. Usually, all of the Kan^(r) colonies wereproperly constructed. This plasmid was referred to as the mini-targetingvector. The floxed Neo gene, together with the homology arms, wasexcised from pBluescript by NotI and SalI digestion, and gel-purified.The purified Neo cassette (150 ng) was co-electroporated along with thegap-repaired subcloned DNA (PL441, 10 ng) into EL350 cells, which hadbeen induced for Red recombination functions by prior growth at 42° C.for 15 minutes, and frozen at −80° C. Transformants were selected onkanamycin plates.

[0387] In one experiment, 84 Kan^(r) colonies were obtained followingelectroporation of induced EL350 cells, while only 6 colonies wereobtained from uninduced cells. All the six colonies were identical tothe original mini-targeting vector, suggesting that they representeduncut plasmid. Plasmids from six of the Kan^(r) colonies from inducedEL350 cells were examined by restriction enzyme digestion to make surethey were the correct recombinants. All 6 colonies gave the expectedrestriction patterns (FIG. 16B, lane 2).

[0388] Not all plasmids in a Kan^(r) cell will carry the Neo cassette.This is especially true for high copy plasmids such as pBluescript sinceone recombinant plasmid molecule will render the cell Kan^(r). The cellswill therefore carry mixtures of targeted and non-targeted plasmidsfollowing recombination. This problem can be reduced if only a smallamount of the gap-repaired subcloned plasmid DNA (1 ng) is used forco-electroporation. Alternatively, the mixed plasmids can beretransformed into DH10B cells and grown on kanamycin plates. Since mosttransformed cells will only receive one plasmid, growth of thetransformed cells on kanamycin plates will select against cells thatreceive non-targeted plasmids, and the surviving colonies will carrypure populations of targeted plasmids.

[0389] Excision of the Neo cassette from the subcloned DNA wasaccomplished by electroporating the targeted plasmid DNA into EL350cells, which had been induced for Cre expression by prior growth inarabinose-containing media for one hour. The electroporated cells wereplated on either ampicillin or kanamycin plates. Cre-mediatedrecombination is highly efficient; therefore, the kanamycin platesusually do not have any colonies. Colonies from the ampicillin plateswere checked for their kanamycin sensitivity and restriction digestionpatterns to make sure that the floxed Neo cassette was properly excised.All 12 Amp^(r) colonies picked for analysis in this experiment werekanamycin sensitive, and contained a single LoxP site at the targetedlocus (FIG. 16B, lane 3).

Example 26 Targeting a Second LoxP Site Downstream of Evi9 Exon 4

[0390] The final step in this example of the construction of thecko-targeting vector is the introduction of a second LoxP into thesubcloned DNA; in this case, downstream of Evi9 exon 4 (FIG. 16A). Oneway to accomplish this task is to again introduce a floxed Neo gene intothe subcloned DNA, and then remove the floxed Neo gene via Crerecombinase, leaving behind a LoxP site at the second targeted locus.This is, however, complicated by the fact that the Neo gene serves asthe selectable marker for gene targeting in ES cells; therefore the Neogene can only be removed after Neo positive ES cells are selected andhomologous recombinants identified. Transient expression of Crerecombinase in ES cells can generate three different excision products:two recombination products are generated by recombination between theLoxP site located upstream of Evi9 exon 4 and the two LoxP sites locateddownstream of Evi9 exon 4, which flank the Neo gene. The third, anddesired recombination product, results from recombination between thetwo-LoxP sites located on either side of the Neo gene. Often, it seemsthat most recombination products are the undesired ones, and in somecases, it can be difficult to obtain ES cells that contain the desiredproduct. Another problem stems from the fact that the Neo gene in apreviously constructed cassette (PGK-Tn5-Kan-bpA) is optimized forexpression in E. coli. Generally, 90% less ES colonies are obtained whenthis cassette is used than when a conventional PGKNeobpA is used.

[0391] To overcome these problems, a new selection cassette (PL451) wasconstructed. PL451 was constructed by introducing a frt site upstream ofNeo, and frt and LoxP sites downstream of Neo, in PGKNeobpA, a selectioncassette that is commonly used for gene targeting in ES cells (FIG.16A). Similar to PL452, a bacterial EM7 promoter was introduced inbetween the PGK promoter and the coding sequence of Neo. This selectioncassette works efficiently in both E. coli and mouse ES cells. fit isthe DNA recognition site for Flp recombinase. DNA located between twofit sites in mouse ES cells can be excised by transient expression of agenetically enhanced Flp recombinase (Flpe) (Buchholz et al., NatBiotechnol 16:657-662 1998), that works well in ES cells. In this case,single frt and single LoxP sites, were left behind at the targeted locus(FIG. 16A). Only one Flpe recombination product is possible, whichensures that all excision products are the correct ones. Alternatively,the PL451 selection cassette can be removed after the conditional alleleis introduced into the mouse germ line by breeding the mice to one ofthe mouse strains that expresses Flpe in the mouse germ line (Rodriguezet al., 2000). Subsequent expression of Cre recombinase will excise theentire DNA between the LoxP sites located on either side of Evi9 exon 4,and create an Evi9 null allele. Cre can be expressed in the mouse germline to create a germ line null allele, or in somatic cells.

[0392] The PL451 selection cassette was introduced into the subclonedDNA in the same manner used to introduce the floxed Neo gene upstream ofEvi9 exon 4. Evi9 exon 4, including both targeted regions, was sequencedto make sure that no undesired mutations were introduced during therecombination process. To functionally test the LoxP and FRT sites inthe targeting vector, the cko-targeting vector plasmid DNA wastransformed into arabinose-induced EL350 and EL250 cells (EL250 cellshave a Flpe gene under the control of the arabinose inducible promoter,PBAD (Lee et al., 2001)), respectively. Cells were plated on ampicillinplates to select for the plasmid. Plasmid DNA was prepared and digestedto confirm the expected recombination patterns (FIG. 16B, lanes 5 and6).

Example 27 Gene Targeting in ES Cells

[0393] The cko-targeting vector was subsequently linearized with NotI,electroporated into CJ7 ES cells, and the transformants selected fortheir G418 and ganciclovir (Ganc) resistance. Homologous recombinationcan occur either upstream or downstream of the LoxP site located 5′ ofEvi9 exon 4. Since a BglII site was introduced along with the upstreamLoxP site, homologous recombinants carrying this LoxP site (the ckoallele) will generate a 18.1 kb (wild type) and a 5.5 kb (mutant) BglIIfragment using a 5′ probe (FIG. 17A). Since an EcoRV site was introducedalong with the selection cassette to the region downstream of exon 4,targeted clones will also have a 6.3 kb EcoRV fragment detected by the3′ probe (FIG. 17A). In one electroporation experiment, 300 G418^(r)Ganc^(r) colonies were obtained following electroporation. Eightycolonies were picked for Southern analysis. Twenty-four out of the 80colonies (30%) had the Evi9 cko allele (FIG. 17B).

[0394] Thus, a rapid and efficient method for generating cko-targetingvectors is disclosed herein. This method relies on E. colirecombineering rather than restriction enzymes and DNA ligases forvector construction (FIG. 18). This method makes use of high copyplasmids rather than BAC DNA to generate the targeting vector, 200-500bp of homology for subcloning (gap repair), and 100-300 bp of homologyfor targeting, rather than the 45-50 bp of homology used in previousexperiments (e.g. see Example 5). By using high copy plasmid DNA forvector construction, the problem caused by Lox sites present in the BACvector backbone is eliminated, and by using longer homology arms, asmany as 10,000 colonies can be obtained from a single subcloningexperiment with only 50-100 ng of retrieving plasmid DNA. In addition,more than 95% of the colonies are correctly constructed. This is incontrast to previous subcloning methods using shorter regions ofhomology. Moreover, using these longer homology arms, targetingfrequencies as high as 1×10⁻² can-be obtained with as little as 100 ngof targeting DNA (i.e., targeting a floxed Neo cassette to a BAC).

[0395] In order to use high copy plasmids such as pBluescript for vectorconstruction, modifications were made in the way the λ Red system wasused. For example, co-electroporation was used to target the floxed Neocassette to the plasmid, instead of introducing the Neo cassette intocells that already carried the plasmid. Induction of the λ Red genesinto cells that carry multiple plasmids can cause the formation ofplasmid complexes due to rolling-circle replication (Feiss et al., Gene17:123-130, 1992). Co-transformation of the Neo cassette and the plasmidminimizes this problem, but still provides a high enough frequency ofhomologous recombination to generate the targeted plasmid.Cre-expressing EL350 cells were also used to excise the floxed Neocassette from the targeted plasmid. When multiple plasmid moleculescontaining LoxP sites are present in a cell expressing Cre,intermolecular recombination between the LoxP sites can occur, resultingin plasmid loss. Electroporation of a small amount of plasmid DNAcontaining the floxed Neo cassette into Cre-expressing EL350 cellsavoids this problem, yet still allows for the efficient excision of theNeo cassette. Two new selection cassettes (loxP-PGK-EM7-NeobpA-loxP andFRT-PGK-EM7-NeobpA-FRT-loxP) were also constructed that worked well inboth E. coli and mouse ES cells. The second selection cassette containstwo frt sites and one LoxP site that flank the selection cassette. Thismakes it possible to remove this selection cassette following homologousrecombination in ES with Flpe recombinase, leaving behind frt-LoxP sitesat the targeted locus.

[0396] Additionally, 200-500 bp homology arms that contain SINE, LINE orshort DNA repeats such as CA repeats have been used for retrieving andtargeting. Efficient recombination was still achieved in all cases. Insome circumstances, longer homology arms can help in avoiding problemscreated by sequencing errors in the public databases, or strainpolymorphisms. This can be of use when modifying human DNA wherepolymorphisms are common. With its high efficiency and reliability, morethan ten cko-targeting vectors have been constructed. Four of thecko-targeting vectors have been introduced into ES cells for homologousrecombination. All four targeting constructs gave rise to highlyefficient gene targeting frequencies in mouse ES cells: the frequency ofcko alleles ranged from 20 to 40% of the G418^(r), Ganc^(r) colonies.

[0397] The most time-consuming step in constructing the cko-targetingvector using this method is in the production of the retrieval vectorand the two mini-targeting vectors. However, since all of the homologyarms used in the construction of these vectors are PCR-amplified fromBAC DNA, only single PCR products are usually obtained, and the PCRproducts can thus be easily purified using spin columns. All six PCRreactions needed to construct a cko vector, including digestion of thePCR products and ligation and transformation, can be done in one day.Typically, it takes less than two weeks to construct a cko-targetingvector using this method, and multiple cko vectors can be generatedsimultaneously. An alternative way to generate longer homology arms forhomologous recombination is by using two-step fusion PCR originallydesigned for enhanced homologous recombination in yeast (Wach, Yeast12:259-265, 1996). With two-step fusion PCR, the two PCR products areamplified that serve as homology regions. Since about 26 base pairs ofselection marker sequences are included in two of the four primers usedto amplify the homology regions, one strand of each of the two PCRproducts can serve as the primer for amplifying the selection marker(Wach, Yeast 12:259-265, 1996).

[0398] By using BACs rather than phage libraries for vectorconstruction, one can precisely choose a genomic region to retrieve forfurther manipulation. Moreover, BACs, and DNA subcloned from BACs intohigh copy plasmids, can be rapidly modified using the methods describedhere to create knock-in mutations and transgene constructs, as well asexpedite the analysis of regulatory elements and functional domains inor near genes via deletion analysis.

[0399] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

1 50 1 20 DNA Artificial sequence primer 1 tgtgacggaa gatcacttcg 20 2 20DNA Artificial sequence primer 2 accagcaata gacataagcg 20 3 20 DNAArtificial sequence primer 3 ctcttgggtt atcaagaggg 20 4 20 DNAArtificial sequence primer 4 actcgacatc ttggttaccg 20 5 21 DNAArtificial sequence primer 5 cattcaaata tgtatccgct c 21 6 20 DNAArtificial sequence primer 6 agagttggta gctcttgatc 20 7 20 DNAArtificial sequence primer 7 tatggacagc aagcgaaccg 20 8 21 DNAArtificial sequence primer 8 tcagaagaac tcgtcaagaa g 21 9 50 DNAArtificial sequence primer 9 gtttgcgcgc agtcagcgat atccattttc gcgaatccggagtgtaagaa 50 10 50 DNA Artificial sequence primer 10 ttcatattgttcagcgacag cttgctgtac ggcaggcacc agctcttccg 50 11 70 DNA Artificialsequence primer 11 aagtcgcggt cggaaccgta ttgcagcagc tttatcatctgccgctggac ggcgcacaaa 60 tcgcgcttaa 70 12 60 DNA Artificial sequenceprimer 12 aacagacacc atggtgcacc tgactcctga ggagaagtct gccgttactgccctgtgggg 60 13 56 DNA Artificial sequence primer 13 ctgcaaggcgattaagttgg gtaacgccag ggttttcgtc aggtggcact ttcggg 56 14 23 DNAArtificial sequence primer 14 ctcagaagaa ctcgtcaaga agg 23 15 35 DNAArtificial sequence primer 15 gtaggtacct cgagaatcgc catcttccag caggc 3516 36 DNA Artificial sequence primer 16 tcgaattttc tgcatccaat ttactgaccgtacacc 36 17 67 DNA Artificial sequence primer 17 gcaagtgtgt cgctgtcgacgagctcgcga gctcggacat gaggttgtct tagacgtcag 60 gtggcac 67 18 69 DNAArtificial sequence primer 18 catagttaag ccagccccga cacccgccaacacccgctga cgcgaacctc acgttaaggg 60 attttggtc 69 19 29 DNA Artificialsequence primer 19 gcaggatcca gtttgctcct ggagcgaca 29 20 22 DNAArtificial sequence primer 20 tgcaggtcga ctctagagga tc 22 21 60 DNAArtificial sequence primer 21 tggcggtgat aatggttgca tgtactaaggaggttgtatg ctcttgggtt atcaagaggg 60 22 60 DNA Artificial sequence primer22 ggcgctgcaa aaattctttg tcgaacaggg tgtctggatc actcgacatc ttggttaccg 6023 61 DNA Artificial sequence primer 23 tggcggtgat aatggttgca tgtactaaggaggttgtatg ctgtgacgga agatcacttc 60 g 61 24 61 DNA Artificial sequenceprimer 24 ggcgctgcaa aaattctttg tcgaacaggg tgtctggatc ctgaggttcttatggctctt 60 g 61 25 60 DNA Artificial sequence primer 25 tggcggtgataatggttgca tgtactaagg aggttgtatg aagcggcatg cataatgtgc 60 26 65 DNAArtificial sequence primer 26 ggcgctgcaa aaattctttg tcgaacagggtgtctggatc ctgtgtccta ctcaggagag 60 cgttc 65 27 63 DNA Artificialsequence primer 27 cgcttcgcgg gacataattt ccgaaatccc agtgtgctgtgagccaagct atcgaattcc 60 gcc 63 28 63 DNA Artificial sequence primer 28gaggctccag gagaatgaga tgttcccgcg ttcaggcaag cgctattcca gaagtagtga 60 gga63 29 79 DNA Artificial sequence primer 29 gcgagcgtgt gagcgcgcgtgggcgcccgg caagccgggg ccatggatta caaggatgac 60 gacgataagg tacaacaga 7930 79 DNA Artificial sequence primer 30 ggccagcaga gcctcagtgt tctccgcgttgttggtctgt tgtaccttat cgtcgtcatc 60 cttgtaatcc atggccccc 79 31 80 DNAArtificial sequence primer 31 ctctccatgc ctgtctgggt gagggtggcccaggggcgat ggctatgaga gaggtcgact 60 tcttagacgt caggtggcac 80 32 79 DNAArtificial sequence primer 32 gcaatgcaga gaagccttgt actgggatgacagagacgga ggggaagagg aggcggccgc 60 gatacgcgag cgaacgtga 79 33 80 DNAArtificial sequence primer 33 gacttctatg acctgtacgg aggggagaagtttgcgacgt gacagagctg gtcgtcgact 60 tcttagacgt caggtggcac 80 34 81 DNAArtificial sequence primer 34 gccccataca cgtaaatgta catagaatcacacagcatca cttctatgga tgcggcggcc 60 gcgatacgcg agcgaacgtg a 81 35 79 DNAArtificial sequence primer 35 catccagtag aacttgggag tgaagctagagccaaggcca tctaagtgac aggcggccgc 60 gatacgcgag cgaacgtga 79 36 23 DNAArtificial sequence primer 36 ctgctggaag atggcgattc tcg 23 37 20 DNAArtificial sequence primer 37 aacagcagga gcggtgagtc 20 38 33 DNAArtificial sequence primer 38 ataagcggcc gctctaatac agactggcac ctg 33 3930 DNA Artificial sequence primer 39 gtcaagcttt aaagagatcc ctgctataaa 3040 30 DNA Artificial sequence primer 40 gtcaagcttc ctgtttccag cgtaggtgaa30 41 30 DNA Artificial sequence primer 41 tctactagtc tcaccacctgtacagtaagt 30 42 34 DNA Artificial sequence primer 42 ataagcggccgcaacaatta gtgtgtttcc agtt 34 43 35 DNA Artificial sequence primer 43gtcgaattca gatctaaatg gggtactgag acaag 35 44 30 DNA Artificial sequenceprimer 44 ataggatcca accaatgaga cagtggcaca 30 45 31 DNA Artificialsequence primer 45 gtcgtcgcac ttattcatgt tccaacaacc a 31 46 33 DNAArtificial sequence primer 46 ataagcggcc gccttaactt agacagcatg tat 33 4729 DNA Artificial sequence primer 47 gtcgaattcg tctgcagagg gttagtcaa 2948 29 DNA Artificial sequence primer 48 ataggatcca gagcagatag cagtgaaaa29 49 30 DNA Artificial sequence primer 49 gtcgtcgcat attacctcacccaatgctag 30 50 34 DNA Artificial sequence primer 50 ataacttcgtataatgtatg ctatacgaag ttat 34

We claim:
 1. A method for generating a vector for conditional knockoutof a gene in a cell, comprising using homologous recombination to inserta nucleic acid encoding a selectable marker flanked by a pair of firstrecombining sites into a first site in a gene in a bacterial artificialchromosome, wherein a vector comprises the bacterial artificialchromosome; excising the nucleic acid encoding the selectable maker witha first recombinase specific for the first recombining sites, wherein asingle first recombining site remains in the gene; using homologousrecombination to insert a nucleic acid encoding a selectable markerflanked by a pair of second recombining sites and a first recombiningsite into a second site in the gene; and excising the nucleic acidencoding the selectable marker with a second recombinase specific forthe second recombining sites, wherein two first recombining sites remainin the gene following excision of the nucleic acid encoding theselectable marker, wherein recombination of the two first recombiningsites produces a nucleic acid sequence that cannot be transcribed toproduce a functional protein, thereby generating the vector forconditional knockout of the gene in the cell.
 2. The method of claim 1,wherein the cell comprises a de-repressible promoter operably linked toa nucleic acid encoding Beta and Exo, and wherein using homologousrecombination comprises activating the de-repressible promoter, therebyinducing the expression of Beta and Exo.
 3. The method of claim 2,wherein either the first recombining sites or the second recombiningsites comprise a LoxP site.
 4. The method of claim 2, wherein the firstrecombining sites comprise a LoxP site, and the second recombining sitescomprise a frt site.
 5. The method of claim 2, wherein the firstrecombining sites comprise a frt site, and the second recombining sitescomprise a LoxP site.
 6. The method of claim 2, wherein using homologousrecombination to insert the nucleic acid encoding the selectable markerflanked by the pair of first recombining sites comprises introducing adouble-stranded vector comprising the nucleic acid encoding theselectable marker flanked by the pair of first recombining sites into ahost cell comprising a nucleic acid sequence encoding Exo, Beta and Gam,operably linked to a de-repressible promoter, wherein the vector furthercomprises a sufficient number of nucleotides homologous to the bacterialartificial chromosome flanking each of the pair of first recombiningsites to achieve homologous recombination; selecting a host cell inwhich homologous recombination has occurred.
 7. The method of claim 2,wherein the cell further comprises an inducible promoter operably linkedto a nucleic acid encoding the first recombinase, and wherein excisingthe nucleic acid encoding the selectable maker comprises inducing theexpression of the first recombinase.
 8. The method of claim 7, whereinthe first recombinase is Cre.
 9. The method of claim 7, wherein thefirst recombinase is Flpe.
 10. The method of claim 7, wherein the cellis a bacterial cell.
 11. The method of claim 7, wherein the cell is aeukaryotic cell.
 12. The method of claim 2, wherein the cell comprisesan inducible promoter operably linked to a nucleic acid encoding thesecond recombinase, and wherein excising the nucleic acid encoding theselectable marker comprises inducing the expression of the secondrecombinase.
 13. The method of claim 1, wherein the selectable markerconfers resistance of the cell to an antibiotic.
 14. A method forgenerating a non-human transgenic animal, the method comprisinglinearizing a vector generated according to the method of claim 2;introducing the vector into an embryonic stem cell, wherein the genecomprising the two first recombining sites is integrated into achromosome of the embryonic stem cell; and producing a transgenic animalfrom the embryonic stem cell.
 15. The method of claim 14, furthercomprising inducing recombination between the first two recombiningsites in the gene, thereby producing a nucleic acid sequence that cannotbe transcribed to produce a functional protein.
 16. The method forgenerating a non-human transgenic animal of claim 14, wherein inducingrecombination between the first two recombining sites in the genecomprises mating the transgenic animal to a second transgenic animal ofthe same species comprising a nucleic acid encoding a recombinaseoperably linked to a conditional promoter; producing an offspringcomprising the gene comprising the two first recombining sites isintegrated into a chromosome and the nucleic acid encoding a recombinaseoperably linked to a conditional promoter; thereby inducingrecombination of the first two recombining sites to produce a nucleicacid sequence that cannot be transcribed to produce the functionalprotein.
 17. The method of claim 14, wherein the non-human transgenicanimal is a transgenic mouse.
 18. A method for introducing a nucleicacid sequence into a gene of interest on an artificial chromosomewithout using drug selction, the method comprising introducing into acell a double-stranded nucleic acid comprising homology arms of at leastforty base pairs in length homologous to the gene of interest, whereinthe homology arms flank a detectable nucleic acid sequence, wherein thedetectable nucleic acid sequence does not encode a polypeptide thatconfers resistance of the cell to a drug, wherein the cell comprises anucleic acid encoding Bet and Exo operably linked to a de-repressiblepromoter; inducing expression of Bet and Exo in the cell, therebyinducing homologous recombination between the homology arms and the geneof interest, and thereby inserting the detectable nucleic acid sequenceinto the gene of interest on the artificial chromosome.
 19. The methodof claim 18, wherein the cell is a bacterial cell.
 20. The method ofclaim 18, wherein the aritificial chromosome is a bacterial artificialchromosome.
 21. The method of claim 17, wherein the de-repressiblepromoter is pL.